Biochemistry, Genetics and Molecular Biology › Molecular Biology

RNA regulation and disease

Works Count: 33530

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This cluster of papers explores the functions and regulation of RNA editing by ADAR deaminases, including its impact on gene regulation, innate immunity, microRNA processing, and the genomic landscape. It covers a wide range of topics such as neurological disorders, cancer, and transcriptome diversity.

Keywords

RNA Editing; ADAR Deaminases; Gene Regulation; Innate Immunity; MicroRNA Processing; Genomic Landscape; Neurological Disorders; Cancer; Transcriptome Diversity; Nucleic Acid Sensing

Glial fibrillary acidic protein: from intermediate filament assembly and gliosis to neurobiomarker

2015-05-11

Zhihui Yang , Kevin Wang

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Evidence That Hepatitis C Virus Resistance to Interferon Is Mediated through Repression of the PKR Protein Kinase by the Nonstructural 5A Protein

1997-04-01

Michael Gale , Marcus J. Korth , Norina Tang +6 more

Brain ischemia and reperfusion: molecular mechanisms of neuronal injury

2000-10-01

Blaine C. White , Jonathon M. Sullivan , Donald J. DeGracia +5 more

Reinitiation involving upstream ORFs regulates ATF4 mRNA translation in mammalian cells

2004-07-26

Krishna M. Vattem , Ronald C. Wek

During cellular stresses, phosphorylation of eukaryotic initiation factor-2 (eIF2) elicits gene expression designed to ameliorate the underlying cellular disturbance. Central to this stress response is the transcriptional regulator activating transcription … During cellular stresses, phosphorylation of eukaryotic initiation factor-2 (eIF2) elicits gene expression designed to ameliorate the underlying cellular disturbance. Central to this stress response is the transcriptional regulator activating transcription factor, ATF4. Here we describe the mechanism regulating ATF4 expression involving the differential contribution of two upstream ORFs (uORFs) in the 5′ leader of the mouse ATF4 mRNA. The 5′ proximal uORF1 is a positive-acting element that facilitates ribosome scanning and reinitiation at downstream coding regions in the ATF4 mRNA. When eIF2-GTP is abundant in nonstressed cells, ribosomes scanning downstream of uORF1 reinitiate at the next coding region, uORF2, an inhibitory element that blocks ATF4 expression. During stress conditions, phosphorylation of eIF2 and the accompanying reduction in the levels of eIF2-GTP increase the time required for the scanning ribosomes to become competent to reinitiate translation. This delayed reinitiation allows for ribosomes to scan through the inhibitory uORF2 and instead reinitiate at the ATF4 -coding region. Increased expression of ATF4 would contribute to the expression of genes involved in remediation of cellular stress damage. These results suggest that the mechanism of translation reinitiation involving uORFs is conserved from yeast to mammals.

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Control of Kinetic Properties of AMPA Receptor Channels by Nuclear RNA Editing

1994-12-09

Hilda Lomelı́ , Johannes Mosbacher , Thorsten Melcher +7 more

AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptor channels mediate the fast component of excitatory postsynaptic currents in the central nervous system. Site-selective nuclear RNA editing controls the calcium permeability of these channels, and … AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptor channels mediate the fast component of excitatory postsynaptic currents in the central nervous system. Site-selective nuclear RNA editing controls the calcium permeability of these channels, and RNA editing at a second site is shown here to affect the kinetic aspects of these channels in rat brain. In three of the four AMPA receptor subunits (GluR-B, -C, and -D), intronic elements determine a codon switch (AGA, arginine, to GGA, glycine) in the primary transcripts in a position termed the R/G site, which immediately precedes the alternatively spliced modules "flip" and "flop." The extent of editing at this site progresses with brain development in a manner specific for subunit and splice form, and edited channels possess faster recovery rates from desensitization.

Widespread A-to-I RNA Editing of Alu-Containing mRNAs in the Human Transcriptome

2004-11-05

A. Athanasiadis , Alexander Rich , Stefan Maas

RNA editing by adenosine deamination generates RNA and protein diversity through the posttranscriptional modification of single nucleotides in RNA sequences. Few mammalian A-to-I edited genes have been identified despite evidence … RNA editing by adenosine deamination generates RNA and protein diversity through the posttranscriptional modification of single nucleotides in RNA sequences. Few mammalian A-to-I edited genes have been identified despite evidence that many more should exist. Here we identify intramolecular pairs of Alu elements as a major target for editing in the human transcriptome. An experimental demonstration in 43 genes was extended by a broader computational analysis of more than 100,000 human mRNAs. We find that 1,445 human mRNAs (1.4%) are subject to RNA editing at more than 14,500 sites, and our data further suggest that the vast majority of pre-mRNAs (greater than 85%) are targeted in introns by the editing machinery. The editing levels of Alu-containing mRNAs correlate with distance and homology between inverted repeats and vary in different tissues. Alu-mediated RNA duplexes targeted by RNA editing are formed intramolecularly, whereas editing due to intermolecular base-pairing appears to be negligible. We present evidence that these editing events can lead to the posttranscriptional creation or elimination of splice signals affecting alternatively spliced Alu-derived exons. The analysis suggests that modification of repetitive elements is a predominant activity for RNA editing with significant implications for cellular gene expression.

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CNS Synaptogenesis Promoted by Glia-Derived Cholesterol

2001-11-09

Daniela H. Mauch , Karl Nägler , Stefan Schumacher +4 more

The molecular mechanisms controlling synaptogenesis in the central nervous system (CNS) are poorly understood. Previous reports showed that a glia-derived factor strongly promotes synapse development in cultures of purified CNS … The molecular mechanisms controlling synaptogenesis in the central nervous system (CNS) are poorly understood. Previous reports showed that a glia-derived factor strongly promotes synapse development in cultures of purified CNS neurons. Here, we identify this factor as cholesterol complexed to apolipoprotein E–containing lipoproteins. CNS neurons produce enough cholesterol to survive and grow, but the formation of numerous mature synapses demands additional amounts that must be provided by glia. Thus, the availability of cholesterol appears to limit synapse development. This may explain the delayed onset of CNS synaptogenesis after glia differentiation and neurobehavioral manifestations of defects in cholesterol or lipoprotein homeostasis.

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Transcriptomic analysis of autistic brain reveals convergent molecular pathology

2011-05-24

Irina Voineagu , Xinchen Wang , Patrick G. Johnston +7 more

GFAP in health and disease

2011-01-09

Jinte Middeldorp , Elly M. Hol

The coxsackievirus and adenovirus receptor is a transmembrane component of the tight junction

2001-12-04

Christopher J. Cohen , Joseph T.C. Shieh , Raymond J. Pickles +3 more

The coxsackievirus and adenovirus receptor (CAR) mediates viral attachment and infection, but its physiologic functions have not been described. In nonpolarized cells, CAR localized to homotypic intercellular contacts, mediated homotypic … The coxsackievirus and adenovirus receptor (CAR) mediates viral attachment and infection, but its physiologic functions have not been described. In nonpolarized cells, CAR localized to homotypic intercellular contacts, mediated homotypic cell aggregation, and recruited the tight junction protein ZO-1 to sites of cell–cell contact. In polarized epithelial cells, CAR and ZO-1 colocalized to tight junctions and could be coprecipitated from cell lysates. CAR expression led to reduced passage of macromolecules and ions across cell monolayers, and soluble CAR inhibited the formation of functional tight junctions. Virus entry into polarized epithelium required disruption of tight junctions. These results indicate that CAR is a component of the tight junction and of the functional barrier to paracellular solute movement. Sequestration of CAR in tight junctions may limit virus infection across epithelial surfaces.

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RNA-binding proteins: modular design for efficient function

2007-05-02

Bradley M. Lunde , Claire Moore , Gabriele Varani

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Analysis of Intron Sequences Reveals Hallmarks of Circular RNA Biogenesis in Animals

2014-12-31

Andranik Ivanov , Sebastian Memczak , Emanuel Wyler +7 more

Circular RNAs (circRNAs) are a large class of animal RNAs. To investigate possible circRNA functions, it is important to understand circRNA biogenesis. Besides human ALU repeats, sequence features that promote … Circular RNAs (circRNAs) are a large class of animal RNAs. To investigate possible circRNA functions, it is important to understand circRNA biogenesis. Besides human ALU repeats, sequence features that promote exon circularization are largely unknown. We experimentally identified circRNAs in C. elegans. Reverse complementary sequences between introns bracketing circRNAs were significantly enriched in comparison to linear controls. By scoring the presence of reverse complementary sequences in human introns, we predicted and experimentally validated circRNAs. We show that introns bracketing circRNAs are highly enriched in RNA editing or hyperediting events. Knockdown of the double-strand RNA-editing enzyme ADAR1 significantly and specifically upregulated circRNA expression. Together, our data support a model of animal circRNA biogenesis in which competing RNA-RNA interactions of introns form larger structures that promote circularization of embedded exons, whereas ADAR1 antagonizes circRNA expression by melting stems within these interactions.

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X-linked dyskeratosis congenita is caused by mutations in a highly conserved gene with putative nucleolar functions

1998-05-01

Nina S. Heiss , S. W. Knight , Tom Vulliamy +5 more

Redirection of Silencing Targets by Adenosine-to-Inosine Editing of miRNAs

2007-02-23

Yukio Kawahara , Boris Zinshteyn , Praveen Sethupathy +3 more

Primary transcripts of certain microRNA (miRNA) genes are subject to RNA editing that converts adenosine to inosine. However, the importance of miRNA editing remains largely undetermined. Here we report that … Primary transcripts of certain microRNA (miRNA) genes are subject to RNA editing that converts adenosine to inosine. However, the importance of miRNA editing remains largely undetermined. Here we report that tissue-specific adenosine-to-inosine editing of miR-376 cluster transcripts leads to predominant expression of edited miR-376 isoform RNAs. One highly edited site is positioned in the middle of the 5′-proximal half “seed” region critical for the hybridization of miRNAs to targets. We provide evidence that the edited miR-376 RNA silences specifically a different set of genes. Repression of phosphoribosyl pyrophosphate synthetase 1, a target of the edited miR-376 RNA and an enzyme involved in the uric-acid synthesis pathway, contributes to tight and tissue-specific regulation of uric-acid levels, revealing a previously unknown role for RNA editing in miRNA-mediated gene silencing.

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Pendred syndrome is caused by mutations in a putative sulphate transporter gene (PDS)

1997-12-01

Lorraine A. Everett , Benjamin Gläser , John C. Beck +7 more

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The eIF2α kinases: their structures and functions

2013-01-25

Neysan Donnelly , Adrienne M. Gorman , Sanjeev Gupta +1 more

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Repression of the human dihydrofolate reductase gene by a non-coding interfering transcript

2007-01-21

Igor Martianov , Aroul Ramadass , Ana Serra Barros +2 more

Molecular cloning and characterization of the human double-stranded RNA-activated protein kinase induced by interferon

1990-07-01

Éliane Meurs , Karen L. Chong , J Galabru +4 more

Inducible and constitutive transcription factors in the mammalian nervous system: control of gene expression by Jun, Fos and Krox, and CREB/ATF proteins

1998-12-01

Thomas Herdegen , J.D. Leah

Relationship of iron to oligondendrocytes and myelination

1996-06-01

James R. Connor , Sharon Menzies

Oligodendrocytes are the predominant iron-containing cells in the brain. Iron-containing oligodendrocytes are found near neuronal cell bodies, along blood vessels, and are particularly abundant within white matter tracts. Iron-positive cells … Oligodendrocytes are the predominant iron-containing cells in the brain. Iron-containing oligodendrocytes are found near neuronal cell bodies, along blood vessels, and are particularly abundant within white matter tracts. Iron-positive cells in white matter are present from birth and eventually reside in defined patches of cells in the adult. These patches of iron-containing cells typically have a blood vessel in their center. Ferritin, the iron storage protein, is also expressed early in development in oligodendrocytes in a regional and cellular pattern similar to that seen for iron. Recently, the functionally distinct subunits of ferritin have been analyzed; only heavy (H)-chain ferritin is found in oligodendrocytes early in development. H-ferritin is associated with high iron utilization and low iron storage. Consistent with the expression of H-ferritin is the expression of transferrin receptors (for iron acquisition) on immature oligodendrocytes. Transferrin protein accumulation and mRNA expression in the brain are both dependent on a viable population of oligodendrocytes and may have an autocrine function to assist oligodendrocytes in iron acquisition. Although apparently the majority of oligodendrocytes in white matter tracts contain ferritin, transferrin, and iron, not all of them do, indicating that there is a subset of oligodendrocytes in white matter tracts. The only known function of oligodendrocytes is myelin production, and both a direct and indirect relationship exists between iron acquisition and myelin production. Iron is directly involved in myelin production as a required co-factor for cholesterol and lipid biosynthesis and indirectly because of its requirement for oxidative metabolism (which occurs in oligodendrocytes at a higher rate than other brain cells). Factors (such as cytokines) and conditions such as iron deficiency may reduce iron acquisition by oligodendrocytes and the susceptibility of oligodendrocytes to oxidative injury may be a result of their iron-rich cytoplasm. Thus, the many known phenomena that decrease oligodendrocyte survival and/or myelin production may mediate their effect through a final common pathway that involves disruptions in iron availability or intracellular management of iron. © 1996 Wiley-Liss, Inc.

Point mutation in an AMPA receptor gene rescues lethality in mice deficient in the RNA-editing enzyme ADAR2

2000-07-01

Miyoko Higuchi , Stefan Maas , Frank N. Single +6 more

PKR; a sentinel kinase for cellular stress

1999-11-01

Bryan R.G. Williams

Translation reinitiation at alternative open reading frames regulates gene expression in an integrated stress response

2004-10-11

Phoebe D. Lu , Heather P. Harding , David Ron

Stress-induced eukaryotic translation initiation factor 2 (eIF2) α phosphorylation paradoxically increases translation of the metazoan activating transcription factor 4 (ATF4), activating the integrated stress response (ISR), a pro-survival gene expression … Stress-induced eukaryotic translation initiation factor 2 (eIF2) α phosphorylation paradoxically increases translation of the metazoan activating transcription factor 4 (ATF4), activating the integrated stress response (ISR), a pro-survival gene expression program. Previous studies implicated the 5′ end of the ATF4 mRNA, with its two conserved upstream ORFs (uORFs), in this translational regulation. Here, we report on mutation analysis of the ATF4 mRNA which revealed that scanning ribosomes initiate translation efficiently at both uORFs and ribosomes that had translated uORF1 efficiently reinitiate translation at downstream AUGs. In unstressed cells, low levels of eIF2α phosphorylation favor early capacitation of such reinitiating ribosomes directing them to the inhibitory uORF2, which precludes subsequent translation of ATF4 and represses the ISR. In stressed cells high levels of eIF2α phosphorylation delays ribosome capacitation and favors reinitiation at ATF4 over the inhibitory uORF2. These features are common to regulated translation of GCN4 in yeast. The metazoan ISR thus resembles the yeast general control response both in its target genes and its mechanistic details.

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An Architectural Role for a Nuclear Noncoding RNA: NEAT1 RNA Is Essential for the Structure of Paraspeckles

2009-02-13

Christine Clemson , John N. Hutchinson , Sergio Andres Sara +4 more

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RNA editing in brain controls a determinant of ion flow in glutamate-gated channels

1991-10-01

Bernd Sommer , Martin Köhler , Rolf Sprengel +1 more

Modulation of microRNA processing and expression through RNA editing by ADAR deaminases

2005-12-20

Weidong Yang , Thimmaiah P. Chendrimada , Qingde Wang +4 more

Inhibition of the Interferon- Inducible Protein Kinase PKR by HCV E2 Protein

1999-07-02

Deborah R. Taylor , Stephanie T. Shi , Patrick R. Romano +2 more

Most isolates of hepatitis C virus (HCV) infections are resistant to interferon, the only available therapy, but the mechanism underlying this resistance has not been defined. Here it is shown … Most isolates of hepatitis C virus (HCV) infections are resistant to interferon, the only available therapy, but the mechanism underlying this resistance has not been defined. Here it is shown that the HCV envelope protein E2 contains a sequence identical with phosphorylation sites of the interferon-inducible protein kinase PKR and the translation initiation factor eIF2α, a target of PKR. E2 inhibited the kinase activity of PKR and blocked its inhibitory effect on protein synthesis and cell growth. This interaction of E2 and PKR may be one mechanism by which HCV circumvents the antiviral effect of interferon.

Phosphorylation of initiation factor eIF-2 and the control of reticulocyte protein synthesis

1977-05-01

Paul J. Farrell , Ken Balkow , Tim Hunt +2 more

An Anthropoid-Specific Locus of Orphan C to U RNA-Editing Enzymes on Chromosome 22

2002-03-01

Adam Jarmuz , Ann Chester , Jayne Bayliss +4 more

Regulation of serotonin-2C receptor G-protein coupling by RNA editing

1997-05-01

Colleen Burns , H. M. Chu , Susan M. Rueter +4 more

Imaging iron stores in the brain using magnetic resonance imaging

2005-01-01

E. Mark Haacke , Yu‐Chung N. Cheng , Michael J. House +7 more

The molecular biology and nomenclature of the activating transcription factor/cAMP responsive element binding family of transcription factors: activating transcription factor proteins and homeostasis

2001-07-01

Tsonwin Hai , Matthew G. Hartman

RNA Editing by Adenosine Deaminases That Act on RNA

2002-06-01

Brenda Bass

▪ Abstract ADARs are RNA editing enzymes that target double-stranded regions of nuclear-encoded RNA and viral RNA. These enzymes are particularly abundant in the nervous system, where they diversify the … ▪ Abstract ADARs are RNA editing enzymes that target double-stranded regions of nuclear-encoded RNA and viral RNA. These enzymes are particularly abundant in the nervous system, where they diversify the information encoded in the genome, for example, by altering codons in mRNAs. The functions of ADARs in known substrates suggest that the enzymes serve to fine-tune and optimize many biological pathways, in ways that we are only starting to imagine. ADARs are also interesting in regard to the remarkable double-stranded structures of their substrates and how enzyme specificity is achieved with little regard to sequence. This review summarizes ongoing investigations of the enzyme family and their substrates, focusing on biological function as well as biochemical mechanism.

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Impact of Protein Kinase PKR in Cell Biology: from Antiviral to Antiproliferative Action

2006-12-01

María Ángel García , Jesús Gil , Iván Ventoso +4 more

SUMMARY The double-stranded RNA-dependent protein kinase PKR is a critical mediator of the antiproliferative and antiviral effects exerted by interferons. Not only is PKR an effector molecule on the cellular … SUMMARY The double-stranded RNA-dependent protein kinase PKR is a critical mediator of the antiproliferative and antiviral effects exerted by interferons. Not only is PKR an effector molecule on the cellular response to double-stranded RNA, but it also integrates signals in response to Toll-like receptor activation, growth factors, and diverse cellular stresses. In this review, we provide a detailed picture on how signaling downstream of PKR unfolds and what are the ultimate consequences for the cell fate. PKR activation affects both transcription and translation. PKR phosphorylation of the alpha subunit of eukaryotic initiation factor 2 results in a blockade on translation initiation. However, PKR cannot avoid the translation of some cellular and viral mRNAs bearing special features in their 5′ untranslated regions. In addition, PKR affects diverse transcriptional factors such as interferon regulatory factor 1, STATs, p53, activating transcription factor 3, and NF-κB. In particular, how PKR triggers a cascade of events involving IKK phosphorylation of IκB and NF-κB nuclear translocation has been intensively studied. At the cellular and organism levels PKR exerts antiproliferative effects, and it is a key antiviral agent. A point of convergence in both effects is that PKR activation results in apoptosis induction. The extent and strength of the antiviral action of PKR are clearly understood by the findings that unrelated viral proteins of animal viruses have evolved to inhibit PKR action by using diverse strategies. The case for the pathological consequences of the antiproliferative action of PKR is less understood, but therapeutic strategies aimed at targeting PKR are beginning to offer promising results.

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Angiogenin cleaves tRNA and promotes stress-induced translational repression

2009-03-30

Satoshi Yamasaki , Pavel Ivanov , Guo‐fu Hu +1 more

Stress-induced phosphorylation of eIF2alpha inhibits global protein synthesis to conserve energy for repair of stress-induced damage. Stress-induced translational arrest is observed in cells expressing a nonphosphorylatable eIF2alpha mutant (S51A), which … Stress-induced phosphorylation of eIF2alpha inhibits global protein synthesis to conserve energy for repair of stress-induced damage. Stress-induced translational arrest is observed in cells expressing a nonphosphorylatable eIF2alpha mutant (S51A), which indicates the existence of an alternative pathway of translational control. In this paper, we show that arsenite, heat shock, or ultraviolet irradiation promotes transfer RNA (tRNA) cleavage and accumulation of tRNA-derived, stress-induced small RNAs (tiRNAs). We show that angiogenin, a secreted ribonuclease, is required for stress-induced production of tiRNAs. Knockdown of angiogenin, but not related ribonucleases, inhibits arsenite-induced tiRNA production and translational arrest. In contrast, knockdown of the angiogenin inhibitor RNH1 enhances tiRNA production and promotes arsenite-induced translational arrest. Moreover, recombinant angiogenin, but not RNase 4 or RNase A, induces tiRNA production and inhibits protein synthesis in the absence of exogenous stress. Finally, transfection of angiogenin-induced tiRNAs promotes phospho-eIF2alpha-independent translational arrest. Our results introduce angiogenin and tiRNAs as components of a phospho-eIF2alpha-independent stress response program.

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Functions and Regulation of RNA Editing by ADAR Deaminases

2010-06-07

Kazuko Nishikura

One type of RNA editing converts adenosines to inosines (A-->I editing) in double-stranded RNA (dsRNA) substrates. A-->I RNA editing is mediated by adenosine deaminase acting on RNA (ADAR) enzymes. A-->I … One type of RNA editing converts adenosines to inosines (A-->I editing) in double-stranded RNA (dsRNA) substrates. A-->I RNA editing is mediated by adenosine deaminase acting on RNA (ADAR) enzymes. A-->I RNA editing of protein-coding sequences of a limited number of mammalian genes results in recoding and subsequent alterations of their functions. However, A-->I RNA editing most frequently targets repetitive RNA sequences located within introns and 5' and 3' untranslated regions (UTRs). Although the biological significance of noncoding RNA editing remains largely unknown, several possibilities, including its role in the control of endogenous short interfering RNAs (esiRNAs), have been proposed. Furthermore, recent studies have revealed that the biogenesis and functions of certain microRNAs (miRNAs) are regulated by the editing of their precursors. Here, I review the recent findings that indicate new functions for A-->I editing in the regulation of noncoding RNAs and for interactions between RNA editing and RNA interference mechanisms.

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A Review of its Symptomatology, Natural History, Pathologic Features, Genetics And Relationship to the Natural Aging Process

1966-05-01

CHARLES J. EPSTKIN , George M. Martin , Amelia L. Schultz +1 more

EPSTKIN, CHARLES J.; MARTIN, GEORGE M.; SCHULTZ, AMELIA L.; MOTULSKYS, ARNO G. Author Information EPSTKIN, CHARLES J.; MARTIN, GEORGE M.; SCHULTZ, AMELIA L.; MOTULSKYS, ARNO G. Author Information

Specific Expression of Activation-induced Cytidine Deaminase (AID), a Novel Member of the RNA-editing Deaminase Family in Germinal Center B Cells

1999-06-01

Masamichi Muramatsu , V.S. Sankaranand , Shrikant Anant +4 more

We have identified a novel gene referred to asactivation-induced deaminase (AID) by subtraction of cDNAs derived from switch-induced and uninduced murine B lymphoma CH12F3-2 cells, more than 80% of which … We have identified a novel gene referred to asactivation-induced deaminase (AID) by subtraction of cDNAs derived from switch-induced and uninduced murine B lymphoma CH12F3-2 cells, more than 80% of which switch exclusively to IgA upon stimulation. The amino acid sequence encoded by AID cDNA is homologous to that of apolipoprotein B (apoB) mRNA-editing enzyme, catalytic polypeptide 1 (APOBEC-1), a type of cytidine deaminase that constitutes a catalytic subunit for the apoB mRNA-editing complex. In vitro experiments using a glutathione S-transferase AID fusion protein revealed significant cytidine deaminase activity that is blocked by tetrahydrouridine and by zinc chelation. However, AID alone did neither demonstrate activity in C to U editing of apoB mRNA nor bind to AU-rich RNA targets. AID mRNA expression is induced in splenic B cells that were activated in vitro or by immunizations with sheep red blood cells. In situ hybridization of immunized spleen sections revealed the restricted expression of AID mRNA in developing germinal centers in which modulation of immunoglobulin gene information through somatic hypermutation and class switch recombination takes place. Taken together, these findings suggest that AID is a new member of the RNA-editing deaminase family and may play a role in genetic events in the germinal center B cell. We have identified a novel gene referred to asactivation-induced deaminase (AID) by subtraction of cDNAs derived from switch-induced and uninduced murine B lymphoma CH12F3-2 cells, more than 80% of which switch exclusively to IgA upon stimulation. The amino acid sequence encoded by AID cDNA is homologous to that of apolipoprotein B (apoB) mRNA-editing enzyme, catalytic polypeptide 1 (APOBEC-1), a type of cytidine deaminase that constitutes a catalytic subunit for the apoB mRNA-editing complex. In vitro experiments using a glutathione S-transferase AID fusion protein revealed significant cytidine deaminase activity that is blocked by tetrahydrouridine and by zinc chelation. However, AID alone did neither demonstrate activity in C to U editing of apoB mRNA nor bind to AU-rich RNA targets. AID mRNA expression is induced in splenic B cells that were activated in vitro or by immunizations with sheep red blood cells. In situ hybridization of immunized spleen sections revealed the restricted expression of AID mRNA in developing germinal centers in which modulation of immunoglobulin gene information through somatic hypermutation and class switch recombination takes place. Taken together, these findings suggest that AID is a new member of the RNA-editing deaminase family and may play a role in genetic events in the germinal center B cell. The germinal center (GC) 1The abbreviations used are: GC, germinal center; CSR, class switch recombination; apoB, apolipoprotein B; APOBEC-1, apoB mRNAediting enzyme, catalytic polypeptide 1; CD40L, CD40 ligand; GST, glutathione S-transferase; THU, tetrahydrouridine; LPS, lipopolysaccharide; RBC, red blood cells; SRBC, sheep RBC; GAPDH, glyceraldehydehyde-3-phosphate dehydrogenase; CHX, cycloheximide; IL, interleukin; TGF, transforming growth factor; RT-PCR, reverse transcription-polymerase chain reaction; kb, kilobase(s).1The abbreviations used are: GC, germinal center; CSR, class switch recombination; apoB, apolipoprotein B; APOBEC-1, apoB mRNAediting enzyme, catalytic polypeptide 1; CD40L, CD40 ligand; GST, glutathione S-transferase; THU, tetrahydrouridine; LPS, lipopolysaccharide; RBC, red blood cells; SRBC, sheep RBC; GAPDH, glyceraldehydehyde-3-phosphate dehydrogenase; CHX, cycloheximide; IL, interleukin; TGF, transforming growth factor; RT-PCR, reverse transcription-polymerase chain reaction; kb, kilobase(s). constitutes a highly specialized microenvironment required for the final maturation step of naive B cells toward antigen-specific memory cells or long-lived plasma cells (1Smith K.G. Light A. Nossal G.J. Tarlinton D.M. EMBO. J. 1997; 16: 2996-3006Crossref PubMed Scopus (333) Google Scholar, 2George J. Claflin L. Semin. Immunol. 1992; 4: 11-17PubMed Google Scholar). Two prominent alterations of immunoglobulin gene information are known to occur in this microenvironment (3Jacob J. Kassir R. Kelsoe G. J. Exp. Med. 1991; 173: 1165-1175Crossref PubMed Scopus (591) Google Scholar, 4Kraal G. Weissman I.L. Butcher E.C. Adv. Exp. Med. Biol. 1985; 186: 145-151PubMed Google Scholar, 5Berek C. Berger A. Apel M. Cell. 1991; 67: 1121-1129Abstract Full Text PDF PubMed Scopus (748) Google Scholar). First, accumulation of massive point mutations in the variable region exon, a process refer to as somatic hypermutation (6Neuberger M.S. Milstein C. Curr. Opin. Immunol. 1995; 7: 248-254Crossref PubMed Scopus (249) Google Scholar), gives rise to affinity maturation of antibody in association with selection of B cells expressing high affinity immunoglobulins on their surface (7Sablitzky F. Wildner G. Rajewsky K. EMBO J. 1985; 4: 345-350Crossref PubMed Scopus (108) Google Scholar, 8Kocks C. Rajewsky K. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8206-8210Crossref PubMed Scopus (147) Google Scholar). Second, class switch recombination (CSR) replaces the exons encoding the heavy chain constant region (9Lorenz M. Radbruch A. Curr. Top. Microbiol. Immunol. 1996; 217: 151-169PubMed Google Scholar), which determines effector functions of the antibody including complement fixation. These two alterations of immunoglobulin gene information are critical for accounting for an effective humoral response to harmful microbes. The molecular mechanisms for these genetic events remain to be elucidated despite intensive study.To dissect the molecular mechanism of class switching, we have isolated a murine B lymphoma clone CH12F3-2 in which CSR from IgM to IgA begins to occur within a few hours after stimulation with IL-4, TGF-β, and CD40L, giving rise to more than 80% IgA+ cells (10Kinoshita K. Tashiro J. Tomita S. Lee Chung-Gi Honjo T. Immunity. 1998; 9: 349-358Abstract Full Text Full Text PDF Scopus (67) Google Scholar, 11Lee C.G. Kondo S. Honjo T. Curr. Biol. 1998; 8: 227-230Abstract Full Text Full Text PDF PubMed Google Scholar, 12Nakamura M. Kondo S. Sugai M. Nazarea M. Imamura S. Honjo T. Int. Immunol. 1996; 8: 193-201Crossref PubMed Scopus (159) Google Scholar). Using CH12F3-2 cells, we have shown that CRS breakpoints are distributed not only within typical repetitive sequences (designated S region) but also in its flanking regions (11Lee C.G. Kondo S. Honjo T. Curr. Biol. 1998; 8: 227-230Abstract Full Text Full Text PDF PubMed Google Scholar). However, the break points were rarely found in the I and C exons, which are separated by the S region, i.e. the intron of germ-line transcripts. Because accumulating evidence indicates that transcription from I to C exon and splicing of the transcripts are essential to CSR (13Hein K. Lorenz M. Siebenkotten B. Petry K. Christine R. Radbruch A. J. Exp. Med. 1998; 188: 2369-2374Crossref PubMed Scopus (124) Google Scholar, 14Lorenz M. Jung S. Radbruch A. Science. 1995; 267: 1825-1828Crossref PubMed Scopus (214) Google Scholar), it is possible that the transcripts are involved directly or indirectly in CSR. We have thus proposed that a complex structure of DNA and RNA, but not S region sequence per se, is recognized for initiation of CSR (11Lee C.G. Kondo S. Honjo T. Curr. Biol. 1998; 8: 227-230Abstract Full Text Full Text PDF PubMed Google Scholar). This idea gained further support with the finding that CSR can take place efficiently even when the Sα region was replaced by the Sε or Sγ1 region in minichromosomal constructs introduced in CH12F3-2 cells upon cytokine stimulation (10Kinoshita K. Tashiro J. Tomita S. Lee Chung-Gi Honjo T. Immunity. 1998; 9: 349-358Abstract Full Text Full Text PDF Scopus (67) Google Scholar).Another type of genetic regulation, RNA-editing, is widely used as a means to create new functional genes from the restricted genome in plants and protozoa (15Scott J. Cell. 1995; 81: 833-836Abstract Full Text PDF PubMed Scopus (101) Google Scholar, 16Simpson L. Thiemann O.H. Cell. 1995; 81: 837-840Abstract Full Text PDF PubMed Scopus (71) Google Scholar). An increasing number of mammalian mRNAs are also known to be edited, including apolipoprotein B (apoB) mRNA, glutamate receptor mRNA, Wilms tumor-1 mRNA, α-galactosidase mRNA, neurofibromatosis type-1 mRNA, and tRNAAsp (17Smith H.C. Sowden M.P. Trends Genet. 1996; 12: 418-424Abstract Full Text PDF PubMed Scopus (50) Google Scholar). Although most of their molecular mechanisms are yet to be elucidated, that of apoB mRNA editing by APOBEC-1(18, 19) is extensively documented. ApoB mRNA editing involves a site-specific C to U deamination of the first base of a CAA codon, encoding glutamine at residue 2153 in apoB100, and produces a UAA in-frame stop codon in apoB48 mRNA (20Navaratnam N. Bhattacharya S. Fujino T. Patel D. Jarmuz A.L. Scott J. Cell. 1995; 81: 187-195Abstract Full Text PDF PubMed Scopus (157) Google Scholar). ApoB100 and apoB48 are translation products of the unedited and edited apoB mRNAs, respectively, and these proteins have completely different physiological functions (21Innerarity T.L. Boren J. Yamanaka S. Olofsson S.O. J. Biol. Chem. 1996; 271: 2353-2356Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). APOBEC-1 requires an auxiliary factor(s) for site-specific RNA editing of apoB mRNA (18Teng B. Burant C.F. Davidson N.O. Science. 1993; 260: 1816-1819Crossref PubMed Scopus (491) Google Scholar, 19Navaratnam N. Morrison J.R. Bhattacharya S. Patel D. Funahashi T. Giannoni F. Teng B.B. Davidson N.O. Scott J. J. Biol. Chem. 1993; 268: 20709-20712Abstract Full Text PDF PubMed Google Scholar). APOBEC-1 itself demonstrates cytidine deaminase activity on a monomeric nucleoside substrate and nonspecific, low affinity binding to AU-rich RNA (19Navaratnam N. Morrison J.R. Bhattacharya S. Patel D. Funahashi T. Giannoni F. Teng B.B. Davidson N.O. Scott J. J. Biol. Chem. 1993; 268: 20709-20712Abstract Full Text PDF PubMed Google Scholar, 20Navaratnam N. Bhattacharya S. Fujino T. Patel D. Jarmuz A.L. Scott J. Cell. 1995; 81: 187-195Abstract Full Text PDF PubMed Scopus (157) Google Scholar, 22MacGinnitie A.J. Anant S. Davidson N.O. J. Biol. Chem. 1995; 270: 14768-14775Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 23Anant S. MacGinnitie A.J. Davidson N.O. J. Biol. Chem. 1995; 270: 14762-14767Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Expression and activity of the auxiliary factors are found not only in organs that carry out apoB mRNA editing but also in those that have no detectable levels of APOBEC-1 or apoB mRNA (18Teng B. Burant C.F. Davidson N.O. Science. 1993; 260: 1816-1819Crossref PubMed Scopus (491) Google Scholar, 19Navaratnam N. Morrison J.R. Bhattacharya S. Patel D. Funahashi T. Giannoni F. Teng B.B. Davidson N.O. Scott J. J. Biol. Chem. 1993; 268: 20709-20712Abstract Full Text PDF PubMed Google Scholar, 24Yamanaka S. Poksay K.S. Balestra M.E. Zeng G.Q. Innerarity T.L. J. Biol. Chem. 1994; 269: 21725-21734Abstract Full Text PDF PubMed Google Scholar). Such widespread expression of the auxiliary factors in the absence of APOBEC-1 implies that the auxiliary factors may be involved in either more general cellular functions or editing of other unknown RNAs. However, little is currently known concerning the identity or activity profile of these other components.In this paper we report isolation of cDNA encoding a novelactivation-induced cytidinedeaminase (AID) that is structurally related to the apoB RNA-editing enzyme, APOBEC-1. The restricted expression and inducibility of AID within B cells in GC suggests that AID may play a role in genetic events in GC.DISCUSSIONTo dissect the molecular mechanism of CSR, we screened subtracted cDNA libraries from nonstimulated and stimulated CH12F3-2 cells on the assumption that induction of trans-acting factors, such as switch recombinase, are required for CSR. In support of this, it was demonstrated that cycloheximide treatment inhibited the formation of looped-out circular DNA in stimulated CH12F3-2 cells (Fig. 1,A and B), indicating the requirement for de novo protein synthesis for CSR to take place.Among four novel genes thus isolated, we have characterized AID, which is specifically induced in GC B cells upon immunization. AID contains the active site for cytidine deaminase and catalyzes deamination of cytidine in vitro (Fig. 3). The inhibitory effect of THU and of zinc chelation on enzyme activity suggests that the deamination process may be similar to that of other cytosine deaminases, including APOBEC-1(19). Phylogenetic analysis revealed that AID is located closer to the RNA-editing deaminase rather than other cytosine deaminases (Fig. 2 B), despite the fact that AID lacks RNA editing activity on an apoB RNA template. Mutagenesis studies indicate that Phe-66, Phe-87, His-61, Glu-63, and Cys-93 in mouse APOBEC-1 are essential for RNA binding (17Smith H.C. Sowden M.P. Trends Genet. 1996; 12: 418-424Abstract Full Text PDF PubMed Scopus (50) Google Scholar, 20Navaratnam N. Bhattacharya S. Fujino T. Patel D. Jarmuz A.L. Scott J. Cell. 1995; 81: 187-195Abstract Full Text PDF PubMed Scopus (157) Google Scholar, 22MacGinnitie A.J. Anant S. Davidson N.O. J. Biol. Chem. 1995; 270: 14768-14775Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 23Anant S. MacGinnitie A.J. Davidson N.O. J. Biol. Chem. 1995; 270: 14762-14767Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). These residues were noted to be conserved in the AID primary structure (Fig. 2 A). However, as noted above, AID was not found to exhibit apoB RNA binding activity. X-ray crystallographic studies on the E. colicytidine deaminase has revealed its three-dimensional structure, which has similarities with the predicted structure of APOBEC-1(34). The cytosine deaminase family can be divided into two groups in the view of the quaternary organization (35Navaratnam N. Fujino T. Bayliss J. Jarmuz A. How A. Richardson N. Somasekaram A. Bhattacharya S. Carter C. Scott J. J. Mol. Biol. 1998; 275: 695-714Crossref PubMed Scopus (124) Google Scholar). E. coli cytidine deaminase and APOBEC-1 have a pseudoactive site domain in the C terminus, which is required to form a homodimer, whereas the other group,e.g. CMP deaminase, lacks such domain and forms homotetramers. AID contains a leucine-rich C terminus, which is shorter than but similar to the pseudoactive site domain of APOBEC-1 (Fig.2 A), indicating that AID belongs to the APOBEC-1/E. coli cytidine deaminase group. The predicted structural similarity of AID to APOBEC-1 implies that AID may be a novel RNA-editing deaminase induced in GC B cells, although it remains to be determined whether AID assembles into either a homodimer or associates into a heteromeric with the auxiliary factors used by APOBEC-1. Further resolution of this issue will require formal identification of the auxiliary factors themselves.AID mRNA was induced in CH12F3-2 cells within 3 h after cytokine stimulation (Fig. 5). The appearance of AID mRNAs and the onset of CSR in CH12F3 cells thus coincide temporally. AID mRNA expression was induced in splenic B cells after in vitrotreatment with LPS and cytokine, which activates naive B cells to initiate CSR (Fig. 6). AID mRNAs were also up-regulated in splenic B cell in vivo when mice were immunized with the T cell-dependent antigen, SRBC. Furthermore, ex vivo experiments (Fig. 7) and in situ hybridization (Fig. 8) studies revealed that AID mRNA was specifically detected in GC B cells, which are competent to perform somatic hypermutation and CSR. In addition, none of the cell lines examined that do not support CSR, including LyD9, BA/F3, 70Z/3, WEHI231, X63, WEHI-3, EL-4, 2B4, F2, P815, L929, NIH3T3, and ST2, were found to express AID mRNA (data not shown). Taken together, these data indicate that AID mRNA is a GC B cell-specific gene induced upon antigen stimulation. Highly restricted expression of the AID gene in GC B cells together with the concordant onset of AID induction and CSR suggest its role in GC function. Furthermore, the possibility of RNA-editing activity on other templates inspires us to speculate that AID may participate in regulatory steps unique to GC function such as somatic hypermutation and CSR. It is of note that the efficiency of CSR in CH12F3-2 cells is increased up to double when cells were stimulated in the presence of THU (data not shown). The physiological function of AID is currently being investigated by gene targeting and transgenic techniques. The germinal center (GC) 1The abbreviations used are: GC, germinal center; CSR, class switch recombination; apoB, apolipoprotein B; APOBEC-1, apoB mRNAediting enzyme, catalytic polypeptide 1; CD40L, CD40 ligand; GST, glutathione S-transferase; THU, tetrahydrouridine; LPS, lipopolysaccharide; RBC, red blood cells; SRBC, sheep RBC; GAPDH, glyceraldehydehyde-3-phosphate dehydrogenase; CHX, cycloheximide; IL, interleukin; TGF, transforming growth factor; RT-PCR, reverse transcription-polymerase chain reaction; kb, kilobase(s).1The abbreviations used are: GC, germinal center; CSR, class switch recombination; apoB, apolipoprotein B; APOBEC-1, apoB mRNAediting enzyme, catalytic polypeptide 1; CD40L, CD40 ligand; GST, glutathione S-transferase; THU, tetrahydrouridine; LPS, lipopolysaccharide; RBC, red blood cells; SRBC, sheep RBC; GAPDH, glyceraldehydehyde-3-phosphate dehydrogenase; CHX, cycloheximide; IL, interleukin; TGF, transforming growth factor; RT-PCR, reverse transcription-polymerase chain reaction; kb, kilobase(s). constitutes a highly specialized microenvironment required for the final maturation step of naive B cells toward antigen-specific memory cells or long-lived plasma cells (1Smith K.G. Light A. Nossal G.J. Tarlinton D.M. EMBO. J. 1997; 16: 2996-3006Crossref PubMed Scopus (333) Google Scholar, 2George J. Claflin L. Semin. Immunol. 1992; 4: 11-17PubMed Google Scholar). Two prominent alterations of immunoglobulin gene information are known to occur in this microenvironment (3Jacob J. Kassir R. Kelsoe G. J. Exp. Med. 1991; 173: 1165-1175Crossref PubMed Scopus (591) Google Scholar, 4Kraal G. Weissman I.L. Butcher E.C. Adv. Exp. Med. Biol. 1985; 186: 145-151PubMed Google Scholar, 5Berek C. Berger A. Apel M. Cell. 1991; 67: 1121-1129Abstract Full Text PDF PubMed Scopus (748) Google Scholar). First, accumulation of massive point mutations in the variable region exon, a process refer to as somatic hypermutation (6Neuberger M.S. Milstein C. Curr. Opin. Immunol. 1995; 7: 248-254Crossref PubMed Scopus (249) Google Scholar), gives rise to affinity maturation of antibody in association with selection of B cells expressing high affinity immunoglobulins on their surface (7Sablitzky F. Wildner G. Rajewsky K. EMBO J. 1985; 4: 345-350Crossref PubMed Scopus (108) Google Scholar, 8Kocks C. Rajewsky K. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8206-8210Crossref PubMed Scopus (147) Google Scholar). Second, class switch recombination (CSR) replaces the exons encoding the heavy chain constant region (9Lorenz M. Radbruch A. Curr. Top. Microbiol. Immunol. 1996; 217: 151-169PubMed Google Scholar), which determines effector functions of the antibody including complement fixation. These two alterations of immunoglobulin gene information are critical for accounting for an effective humoral response to harmful microbes. The molecular mechanisms for these genetic events remain to be elucidated despite intensive study. To dissect the molecular mechanism of class switching, we have isolated a murine B lymphoma clone CH12F3-2 in which CSR from IgM to IgA begins to occur within a few hours after stimulation with IL-4, TGF-β, and CD40L, giving rise to more than 80% IgA+ cells (10Kinoshita K. Tashiro J. Tomita S. Lee Chung-Gi Honjo T. Immunity. 1998; 9: 349-358Abstract Full Text Full Text PDF Scopus (67) Google Scholar, 11Lee C.G. Kondo S. Honjo T. Curr. Biol. 1998; 8: 227-230Abstract Full Text Full Text PDF PubMed Google Scholar, 12Nakamura M. Kondo S. Sugai M. Nazarea M. Imamura S. Honjo T. Int. Immunol. 1996; 8: 193-201Crossref PubMed Scopus (159) Google Scholar). Using CH12F3-2 cells, we have shown that CRS breakpoints are distributed not only within typical repetitive sequences (designated S region) but also in its flanking regions (11Lee C.G. Kondo S. Honjo T. Curr. Biol. 1998; 8: 227-230Abstract Full Text Full Text PDF PubMed Google Scholar). However, the break points were rarely found in the I and C exons, which are separated by the S region, i.e. the intron of germ-line transcripts. Because accumulating evidence indicates that transcription from I to C exon and splicing of the transcripts are essential to CSR (13Hein K. Lorenz M. Siebenkotten B. Petry K. Christine R. Radbruch A. J. Exp. Med. 1998; 188: 2369-2374Crossref PubMed Scopus (124) Google Scholar, 14Lorenz M. Jung S. Radbruch A. Science. 1995; 267: 1825-1828Crossref PubMed Scopus (214) Google Scholar), it is possible that the transcripts are involved directly or indirectly in CSR. We have thus proposed that a complex structure of DNA and RNA, but not S region sequence per se, is recognized for initiation of CSR (11Lee C.G. Kondo S. Honjo T. Curr. Biol. 1998; 8: 227-230Abstract Full Text Full Text PDF PubMed Google Scholar). This idea gained further support with the finding that CSR can take place efficiently even when the Sα region was replaced by the Sε or Sγ1 region in minichromosomal constructs introduced in CH12F3-2 cells upon cytokine stimulation (10Kinoshita K. Tashiro J. Tomita S. Lee Chung-Gi Honjo T. Immunity. 1998; 9: 349-358Abstract Full Text Full Text PDF Scopus (67) Google Scholar). Another type of genetic regulation, RNA-editing, is widely used as a means to create new functional genes from the restricted genome in plants and protozoa (15Scott J. Cell. 1995; 81: 833-836Abstract Full Text PDF PubMed Scopus (101) Google Scholar, 16Simpson L. Thiemann O.H. Cell. 1995; 81: 837-840Abstract Full Text PDF PubMed Scopus (71) Google Scholar). An increasing number of mammalian mRNAs are also known to be edited, including apolipoprotein B (apoB) mRNA, glutamate receptor mRNA, Wilms tumor-1 mRNA, α-galactosidase mRNA, neurofibromatosis type-1 mRNA, and tRNAAsp (17Smith H.C. Sowden M.P. Trends Genet. 1996; 12: 418-424Abstract Full Text PDF PubMed Scopus (50) Google Scholar). Although most of their molecular mechanisms are yet to be elucidated, that of apoB mRNA editing by APOBEC-1(18, 19) is extensively documented. ApoB mRNA editing involves a site-specific C to U deamination of the first base of a CAA codon, encoding glutamine at residue 2153 in apoB100, and produces a UAA in-frame stop codon in apoB48 mRNA (20Navaratnam N. Bhattacharya S. Fujino T. Patel D. Jarmuz A.L. Scott J. Cell. 1995; 81: 187-195Abstract Full Text PDF PubMed Scopus (157) Google Scholar). ApoB100 and apoB48 are translation products of the unedited and edited apoB mRNAs, respectively, and these proteins have completely different physiological functions (21Innerarity T.L. Boren J. Yamanaka S. Olofsson S.O. J. Biol. Chem. 1996; 271: 2353-2356Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). APOBEC-1 requires an auxiliary factor(s) for site-specific RNA editing of apoB mRNA (18Teng B. Burant C.F. Davidson N.O. Science. 1993; 260: 1816-1819Crossref PubMed Scopus (491) Google Scholar, 19Navaratnam N. Morrison J.R. Bhattacharya S. Patel D. Funahashi T. Giannoni F. Teng B.B. Davidson N.O. Scott J. J. Biol. Chem. 1993; 268: 20709-20712Abstract Full Text PDF PubMed Google Scholar). APOBEC-1 itself demonstrates cytidine deaminase activity on a monomeric nucleoside substrate and nonspecific, low affinity binding to AU-rich RNA (19Navaratnam N. Morrison J.R. Bhattacharya S. Patel D. Funahashi T. Giannoni F. Teng B.B. Davidson N.O. Scott J. J. Biol. Chem. 1993; 268: 20709-20712Abstract Full Text PDF PubMed Google Scholar, 20Navaratnam N. Bhattacharya S. Fujino T. Patel D. Jarmuz A.L. Scott J. Cell. 1995; 81: 187-195Abstract Full Text PDF PubMed Scopus (157) Google Scholar, 22MacGinnitie A.J. Anant S. Davidson N.O. J. Biol. Chem. 1995; 270: 14768-14775Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 23Anant S. MacGinnitie A.J. Davidson N.O. J. Biol. Chem. 1995; 270: 14762-14767Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Expression and activity of the auxiliary factors are found not only in organs that carry out apoB mRNA editing but also in those that have no detectable levels of APOBEC-1 or apoB mRNA (18Teng B. Burant C.F. Davidson N.O. Science. 1993; 260: 1816-1819Crossref PubMed Scopus (491) Google Scholar, 19Navaratnam N. Morrison J.R. Bhattacharya S. Patel D. Funahashi T. Giannoni F. Teng B.B. Davidson N.O. Scott J. J. Biol. Chem. 1993; 268: 20709-20712Abstract Full Text PDF PubMed Google Scholar, 24Yamanaka S. Poksay K.S. Balestra M.E. Zeng G.Q. Innerarity T.L. J. Biol. Chem. 1994; 269: 21725-21734Abstract Full Text PDF PubMed Google Scholar). Such widespread expression of the auxiliary factors in the absence of APOBEC-1 implies that the auxiliary factors may be involved in either more general cellular functions or editing of other unknown RNAs. However, little is currently known concerning the identity or activity profile of these other components. In this paper we report isolation of cDNA encoding a novelactivation-induced cytidinedeaminase (AID) that is structurally related to the apoB RNA-editing enzyme, APOBEC-1. The restricted expression and inducibility of AID within B cells in GC suggests that AID may play a role in genetic events in GC. DISCUSSIONTo dissect the molecular mechanism of CSR, we screened subtracted cDNA libraries from nonstimulated and stimulated CH12F3-2 cells on the assumption that induction of trans-acting factors, such as switch recombinase, are required for CSR. In support of this, it was demonstrated that cycloheximide treatment inhibited the formation of looped-out circular DNA in stimulated CH12F3-2 cells (Fig. 1,A and B), indicating the requirement for de novo protein synthesis for CSR to take place.Among four novel genes thus isolated, we have characterized AID, which is specifically induced in GC B cells upon immunization. AID contains the active site for cytidine deaminase and catalyzes deamination of cytidine in vitro (Fig. 3). The inhibitory effect of THU and of zinc chelation on enzyme activity suggests that the deamination process may be similar to that of other cytosine deaminases, including APOBEC-1(19). Phylogenetic analysis revealed that AID is located closer to the RNA-editing deaminase rather than other cytosine deaminases (Fig. 2 B), despite the fact that AID lacks RNA editing activity on an apoB RNA template. Mutagenesis studies indicate that Phe-66, Phe-87, His-61, Glu-63, and Cys-93 in mouse APOBEC-1 are essential for RNA binding (17Smith H.C. Sowden M.P. Trends Genet. 1996; 12: 418-424Abstract Full Text PDF PubMed Scopus (50) Google Scholar, 20Navaratnam N. Bhattacharya S. Fujino T. Patel D. Jarmuz A.L. Scott J. Cell. 1995; 81: 187-195Abstract Full Text PDF PubMed Scopus (157) Google Scholar, 22MacGinnitie A.J. Anant S. Davidson N.O. J. Biol. Chem. 1995; 270: 14768-14775Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 23Anant S. MacGinnitie A.J. Davidson N.O. J. Biol. Chem. 1995; 270: 14762-14767Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). These residues were noted to be conserved in the AID primary structure (Fig. 2 A). However, as noted above, AID was not found to exhibit apoB RNA binding activity. X-ray crystallographic studies on the E. colicytidine deaminase has revealed its three-dimensional structure, which has similarities with the predicted structure of APOBEC-1(34). The cytosine deaminase family can be divided into two groups in the view of the quaternary organization (35Navaratnam N. Fujino T. Bayliss J. Jarmuz A. How A. Richardson N. Somasekaram A. Bhattacharya S. Carter C. Scott J. J. Mol. Biol. 1998; 275: 695-714Crossref PubMed Scopus (124) Google Scholar). E. coli cytidine deaminase and APOBEC-1 have a pseudoactive site domain in the C terminus, which is required to form a homodimer, whereas the other group,e.g. CMP deaminase, lacks such domain and forms homotetramers. AID contains a leucine-rich C terminus, which is shorter than but similar to the pseudoactive site domain of APOBEC-1 (Fig.2 A), indicating that AID belongs to the APOBEC-1/E. coli cytidine deaminase group. The predicted structural similarity of AID to APOBEC-1 implies that AID may be a novel RNA-editing deaminase induced in GC B cells, although it remains to be determined whether AID assembles into either a homodimer or associates into a heteromeric with the auxiliary factors used by APOBEC-1. Further resolution of this issue will require formal identification of the auxiliary factors themselves.AID mRNA was induced in CH12F3-2 cells within 3 h after cytokine stimulation (Fig. 5). The appearance of AID mRNAs and the onset of CSR in CH12F3 cells thus coincide temporally. AID mRNA expression was induced in splenic B cells after in vitrotreatment with LPS and cytokine, which activates naive B cells to initiate CSR (Fig. 6). AID mRNAs were also up-regulated in splenic B cell in vivo when mice were immunized with the T cell-dependent antigen, SRBC. Furthermore, ex vivo experiments (Fig. 7) and in situ hybridization (Fig. 8) studies revealed that AID mRNA was specifically detected in GC B cells, which are competent to perform somatic hypermutation and CSR. In addition, none of the cell lines examined that do not support CSR, including LyD9, BA/F3, 70Z/3, WEHI231, X63, WEHI-3, EL-4, 2B4, F2, P815, L929, NIH3T3, and ST2, were found to express AID mRNA (data not shown). Taken together, these data indicate that AID mRNA is a GC B cell-specific gene induced upon antigen stimulation. Highly restricted expression of the AID gene in GC B cells together with the concordant onset of AID induction and CSR suggest its role in GC function. Furthermore, the possibility of RNA-editing activity on other templates inspires us to speculate that AID may participate in regulatory steps unique to GC function such as somatic hypermutation and CSR. It is of note that the efficiency of CSR in CH12F3-2 cells is increased up to double when cells were stimulated in the presence of THU (data not shown). The physiological function of AID is currently being investigated by gene targeting and transgenic techniques. To dissect the molecular mechanism of CSR, we screened subtracted cDNA libraries from nonstimulated and stimulated CH12F3-2 cells on the assumption that induction of trans-acting factors, such as switch recombinase, are required for CSR. In support of this, it was demonstrated that cycloheximide treatment inhibited the formation of looped-out circular DNA in stimulated CH12F3-2 cells (Fig. 1,A and B), indicating the requirement for de novo protein synthesis for CSR to take place. Among four novel genes thus isolated, we have characterized AID, which is specifically induced in GC B cells upon immunization. AID contains the active site for cytidine deaminase and catalyzes deamination of cytidine in vitro (Fig. 3). The inhibitory effect of THU and of zinc chelation on enzyme activity suggests that the deamination process may be similar to that of other cytosine deaminases, including APOBEC-1(19). Phylogenetic analysis revealed that AID is located closer to the RNA-editing deaminase rather than other cytosine deaminases (Fig. 2 B), despite the fact that AID lacks RNA editing activity on an apoB RNA template. Mutagenesis studies indicate that Phe-66, Phe-87, His-61, Glu-63, and Cys-93 in mouse APOBEC-1 are essential for RNA binding (17Smith H.C. Sowden M.P. Trends Genet. 1996; 12: 418-424Abstract Full Text PDF PubMed Scopus (50) Google Scholar, 20Navaratnam N. Bhattacharya S. Fujino T. Patel D. Jarmuz A.L. Scott J. Cell. 1995; 81: 187-195Abstract Full Text PDF PubMed Scopus (157) Google Scholar, 22MacGinnitie A.J. Anant S. Davidson N.O. J. Biol. Chem. 1995; 270: 14768-14775Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 23Anant S. MacGinnitie A.J. Davidson N.O. J. Biol. Chem. 1995; 270: 14762-14767Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). These residues were noted to be conserved in the AID primary structure (Fig. 2 A). However, as noted above, AID was not found to exhibit apoB RNA binding activity. X-ray crystallographic studies on the E. colicytidine deaminase has revealed its three-dimensional structure, which has similarities with the predicted structure of APOBEC-1(34). The cytosine deaminase family can be divided into two groups in the view of the quaternary organization (35Navaratnam N. Fujino T. Bayliss J. Jarmuz A. How A. Richardson N. Somasekaram A. Bhattacharya S. Carter C. Scott J. J. Mol. Biol. 1998; 275: 695-714Crossref PubMed Scopus (124) Google Scholar). E. coli cytidine deaminase and APOBEC-1 have a pseudoactive site domain in the C terminus, which is required to form a homodimer, whereas the other group,e.g. CMP deaminase, lacks such domain and forms homotetramers. AID contains a leucine-rich C terminus, which is shorter than but similar to the pseudoactive site domain of APOBEC-1 (Fig.2 A), indicating that AID belongs to the APOBEC-1/E. coli cytidine deaminase group. The predicted structural similarity of AID to APOBEC-1 implies that AID may be a novel RNA-editing deaminase induced in GC B cells, although it remains to be determined whether AID assembles into either a homodimer or associates into a heteromeric with the auxiliary factors used by APOBEC-1. Further resolution of this issue will require formal identification of the auxiliary factors themselves. AID mRNA was induced in CH12F3-2 cells within 3 h after cytokine stimulation (Fig. 5). The appearance of AID mRNAs and the onset of CSR in CH12F3 cells thus coincide temporally. AID mRNA expression was induced in splenic B cells after in vitrotreatment with LPS and cytokine, which activates naive B cells to initiate CSR (Fig. 6). AID mRNAs were also up-regulated in splenic B cell in vivo when mice were immunized with the T cell-dependent antigen, SRBC. Furthermore, ex vivo experiments (Fig. 7) and in situ hybridization (Fig. 8) studies revealed that AID mRNA was specifically detected in GC B cells, which are competent to perform somatic hypermutation and CSR. In addition, none of the cell lines examined that do not support CSR, including LyD9, BA/F3, 70Z/3, WEHI231, X63, WEHI-3, EL-4, 2B4, F2, P815, L929, NIH3T3, and ST2, were found to express AID mRNA (data not shown). Taken together, these data indicate that AID mRNA is a GC B cell-specific gene induced upon antigen stimulation. Highly restricted expression of the AID gene in GC B cells together with the concordant onset of AID induction and CSR suggest its role in GC function. Furthermore, the possibility of RNA-editing activity on other templates inspires us to speculate that AID may participate in regulatory steps unique to GC function such as somatic hypermutation and CSR. It is of note that the efficiency of CSR in CH12F3-2 cells is increased up to double when cells were stimulated in the presence of THU (data not shown). The physiological function of AID is currently being investigated by gene targeting and transgenic techniques. We thank Drs. H. Ikuta S. Minoguchi, and K. Kuroda for discussion and suggestions. We also thank M. Yamamoto and T. Tabuchi for their technical assistance and Y. Takahashi and T. Tanaka for secretarial help.

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2015-07-24

Brian Liddicoat , Robert Piskol , Alistair M. Chalk +6 more

Adenosine-to-inosine (A-to-I) editing is a highly prevalent posttranscriptional modification of RNA, mediated by ADAR (adenosine deaminase acting on RNA) enzymes. In addition to RNA editing, additional functions have been proposed … Adenosine-to-inosine (A-to-I) editing is a highly prevalent posttranscriptional modification of RNA, mediated by ADAR (adenosine deaminase acting on RNA) enzymes. In addition to RNA editing, additional functions have been proposed for ADAR1. To determine the specific role of RNA editing by ADAR1, we generated mice with an editing-deficient knock-in mutation (Adar1(E861A), where E861A denotes Glu(861)→Ala(861)). Adar1(E861A/E861A) embryos died at ~E13.5 (embryonic day 13.5), with activated interferon and double-stranded RNA (dsRNA)-sensing pathways. Genome-wide analysis of the in vivo substrates of ADAR1 identified clustered hyperediting within long dsRNA stem loops within 3' untranslated regions of endogenous transcripts. Finally, embryonic death and phenotypes of Adar1(E861A/E861A) were rescued by concurrent deletion of the cytosolic sensor of dsRNA, MDA5. A-to-I editing of endogenous dsRNA is the essential function of ADAR1, preventing the activation of the cytosolic dsRNA response by endogenous transcripts.

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Antiviral Actions of Interferons

2001-10-01

Charles E. Samuel

SUMMARY Tremendous progress has been made in understanding the molecular basis of the antiviral actions of interferons (IFNs), as well as strategies evolved by viruses to antagonize the actions of … SUMMARY Tremendous progress has been made in understanding the molecular basis of the antiviral actions of interferons (IFNs), as well as strategies evolved by viruses to antagonize the actions of IFNs. Furthermore, advances made while elucidating the IFN system have contributed significantly to our understanding in multiple areas of virology and molecular cell biology, ranging from pathways of signal transduction to the biochemical mechanisms of transcriptional and translational control to the molecular basis of viral pathogenesis. IFNs are approved therapeutics and have moved from the basic research laboratory to the clinic. Among the IFN-induced proteins important in the antiviral actions of IFNs are the RNA-dependent protein kinase (PKR), the 2′,5′-oligoadenylate synthetase (OAS) and RNase L, and the Mx protein GTPases. Double-stranded RNA plays a central role in modulating protein phosphorylation and RNA degradation catalyzed by the IFN-inducible PKR kinase and the 2′-5′-oligoadenylate-dependent RNase L, respectively, and also in RNA editing by the IFN-inducible RNA-specific adenosine deaminase (ADAR1). IFN also induces a form of inducible nitric oxide synthase (iNOS2) and the major histocompatibility complex class I and II proteins, all of which play important roles in immune response to infections. Several additional genes whose expression profiles are altered in response to IFN treatment and virus infection have been identified by microarray analyses. The availability of cDNA and genomic clones for many of the components of the IFN system, including IFN-α, IFN-β, and IFN-γ, their receptors, Jak and Stat and IRF signal transduction components, and proteins such as PKR, 2′,5′-OAS, Mx, and ADAR, whose expression is regulated by IFNs, has permitted the generation of mutant proteins, cells that overexpress different forms of the proteins, and animals in which their expression has been disrupted by targeted gene disruption. The use of these IFN system reagents, both in cell culture and in whole animals, continues to provide important contributions to our understanding of the virus-host interaction and cellular antiviral response.

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Identification and Characterization of Pancreatic Eukaryotic Initiation Factor 2 α-Subunit Kinase, PEK, Involved in Translational Control

1998-12-01

Yuguang Shi , Krishna M. Vattem , Ruchira Sood +4 more

In response to various environmental stresses, eukaryotic cells down-regulate protein synthesis by phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF-2α). In mammals, the phosphorylation was shown … In response to various environmental stresses, eukaryotic cells down-regulate protein synthesis by phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF-2α). In mammals, the phosphorylation was shown to be carried out by eIF-2α kinases PKR and HRI. We report the identification and characterization of a cDNA from rat pancreatic islet cells that encodes a new related kinase, which we term pancreatic eIF-2α kinase, or PEK. In addition to a catalytic domain with sequence and structural features conserved among eIF-2α kinases, PEK contains a distinctive amino-terminal region 550 residues in length. Using recombinant PEK produced in Escherichia coli or Sf-9 insect cells, we demonstrate that PEK is autophosphorylated on both serine and threonine residues and that the recombinant enzyme can specifically phosphorylate eIF-2α on serine-51. Northern blot analyses indicate that PEK mRNA is expressed in all tissues examined, with highest levels in pancreas cells. Consistent with our mRNA assays, PEK activity was predominantly detected in pancreas and pancreatic islet cells. The regulatory role of PEK in protein synthesis was demonstrated both in vitro and in vivo. The addition of recombinant PEK to reticulocyte lysates caused a dose-dependent inhibition of translation. In the Saccharomyces model system, PEK functionally substituted for the endogenous yeast eIF-2α kinase, GCN2, by a process requiring the serine-51 phosphorylation site in eIF-2α. We also identified PEK homologs from both Caenorhabditis elegans and the puffer fish Fugu rubripes, suggesting that this eIF-2α kinase plays an important role in translational control from nematodes to mammals.

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Class Switch Recombination and Hypermutation Require Activation-Induced Cytidine Deaminase (AID), a Potential RNA Editing Enzyme

2000-09-01

Masamichi Muramatsu , Kazuo Kinoshita , Sidonia Fagarasan +3 more

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RNAi

2000-03-01

Phillip D. Zamore , Thomas Tuschl , Phillip A. Sharp +1 more

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A-to-I editing of coding and non-coding RNAs by ADARs

2015-12-09

Kazuko Nishikura

Astrocytes: a central element in neurological diseases

2015-12-15

Milos Pekny , Marcela Pekna , Albee Messing +6 more

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RNA editing with CRISPR-Cas13

2017-10-25

David Cox , Jonathan S. Gootenberg , Omar O. Abudayyeh +4 more

Nucleic acid editing holds promise for treating genetic disease, particularly at the RNA level, where disease-relevant sequences can be rescued to yield functional protein products. Type VI CRISPR-Cas systems contain … Nucleic acid editing holds promise for treating genetic disease, particularly at the RNA level, where disease-relevant sequences can be rescued to yield functional protein products. Type VI CRISPR-Cas systems contain the programmable single-effector RNA-guided ribonuclease Cas13. We profiled type VI systems in order to engineer a Cas13 ortholog capable of robust knockdown and demonstrated RNA editing by using catalytically inactive Cas13 (dCas13) to direct adenosine-to-inosine deaminase activity by ADAR2 (adenosine deaminase acting on RNA type 2) to transcripts in mammalian cells. This system, referred to as RNA Editing for Programmable A to I Replacement (REPAIR), which has no strict sequence constraints, can be used to edit full-length transcripts containing pathogenic mutations. We further engineered this system to create a high-specificity variant and minimized the system to facilitate viral delivery. REPAIR presents a promising RNA-editing platform with broad applicability for research, therapeutics, and biotechnology.

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Deciphering miRNAs’ Action through miRNA Editing

2019-12-11

Marta Correia de Sousa , Monika Gjorgjieva , Dobrochna Dolicka +2 more

MicroRNAs (miRNAs) are small non-coding RNAs with the capability of modulating gene expression at the post-transcriptional level either by inhibiting messenger RNA (mRNA) translation or by promoting mRNA degradation. The … MicroRNAs (miRNAs) are small non-coding RNAs with the capability of modulating gene expression at the post-transcriptional level either by inhibiting messenger RNA (mRNA) translation or by promoting mRNA degradation. The outcome of a myriad of physiological processes and pathologies, including cancer, cardiovascular and metabolic diseases, relies highly on miRNAs. However, deciphering the precise roles of specific miRNAs in these pathophysiological contexts is challenging due to the high levels of complexity of their actions. Indeed, regulation of mRNA expression by miRNAs is frequently cell/organ specific; highly dependent on the stress and metabolic status of the organism; and often poorly correlated with miRNA expression levels. Such biological features of miRNAs suggest that various regulatory mechanisms control not only their expression, but also their activity and/or bioavailability. Several mechanisms have been described to modulate miRNA action, including genetic polymorphisms, methylation of miRNA promoters, asymmetric miRNA strand selection, interactions with RNA-binding proteins (RBPs) or other coding/non-coding RNAs. Moreover, nucleotide modifications (A-to-I or C-to-U) within the miRNA sequences at different stages of their maturation are also critical for their functionality. This regulatory mechanism called "RNA editing" involves specific enzymes of the adenosine/cytidine deaminase family, which trigger single nucleotide changes in primary miRNAs. These nucleotide modifications greatly influence a miRNA's stability, maturation and activity by changing its specificity towards target mRNAs. Understanding how editing events impact miRNA's ability to regulate stress responses in cells and organs, or the development of specific pathologies, e.g., metabolic diseases or cancer, should not only deepen our knowledge of molecular mechanisms underlying complex diseases, but can also facilitate the design of new therapeutic approaches based on miRNA targeting. Herein, we will discuss the current knowledge on miRNA editing and how this mechanism regulates miRNA biogenesis and activity.

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Systematic identification of abundant A-to-I editing sites in the human transcriptome

2004-07-18

Erez Y. Levanon , Eli Eisenberg , Rodrigo Yelin +7 more

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The expanding repertoire of ESCRT functions in cell biology and disease

2025-06-25

James H. Hurley , Alyssa N. Coyne , Marta Miączyńska +1 more | Nature

PADI4-mediated citrullination of histone H3 stimulates HIV-1 transcription

2025-06-25

Luca Love , Bianca B. Jütte , Birgitta Lindqvist +4 more | Nature Communications

FRRS1L variants and ferriheme overload drive hyperpigmentation and systemic Iron overload in lanping black bone sheep

2025-06-24

Deping Han , Yuanyuan Zhang , Weidong Deng +7 more | Cell & Bioscience

A Novel Pathogenic Variant Identified in HIKESHI-Related Hypomyelinating Leukodystrophy Disrupts Heat Shock Response in iPSCs

2025-06-24

Mahmood Ali Saleh , Maria Boichuck , Aner Ottolenghi +7 more | International Journal of Molecular Sciences

HIKESHI-related hypomyelinating leukodystrophy (HHL) is a life-threatening disorder caused by homozygous pathogenic variants in HIKESHI. Symptoms include infantile onset progressive spastic dystonic quadriplegia, nystagmus, failure to thrive, diffused hypomyelination, and … HIKESHI-related hypomyelinating leukodystrophy (HHL) is a life-threatening disorder caused by homozygous pathogenic variants in HIKESHI. Symptoms include infantile onset progressive spastic dystonic quadriplegia, nystagmus, failure to thrive, diffused hypomyelination, and severe morbidity or death following febrile illness. V54L variants in HIKESHI are particularly prevalent within the Ashkenazi Jewish population. Here, we identified a novel P78S disease-causing variant in HIKESHI in a patient of Christian Arab origin, presenting with clinical and radiologic features characteristic of HHL. In silico analysis suggests that the mutated residue may affect the HIKESHI protein’s dimerization domain. We generated a comprehensive set of induced pluripotent stem cells (iPSCs) from the index case and two additional HHL patients. To investigate mechanisms potentially linked to febrile illness in HHL, we used these cells to study the heat shock (HS) response. HHL-iPSCs showed dramatically decreased levels of HIKESHI compared with healthy controls following HS. In addition, they exhibited increased HSP70 mRNA levels in response to HS, suggesting an increased sensitivity. HHL-iPSCs had impaired HSP70 translocation to the nucleus. Our results provide a human-relevant model for HHL.

Rapid and Cost-Effective Digital Quantification of RNA Editing and Maturation in Organelle Transcripts

2025-06-24

Zhihua Hua | bioRxiv (Cold Spring Harbor Laboratory)

RNA editing and maturation are critical regulatory mechanisms in plant organelles, yet their quantification remains technically challenging. Traditional Sanger sequencing lacks sensitivity and reproducibility, whereas advanced next-generation sequencing (NGS) approaches, … RNA editing and maturation are critical regulatory mechanisms in plant organelles, yet their quantification remains technically challenging. Traditional Sanger sequencing lacks sensitivity and reproducibility, whereas advanced next-generation sequencing (NGS) approaches, such as rRNA-depleted RNA-seq or targeted amplicon-seq, involve high costs, complex workflows, and limited accessibility. To address these limitations, we developed a rapid and cost-effective long-read sequencing approach, termed premium PCR sequencing, for digital quantification of RNA-editing and intron retention events in targeted chloroplast transcripts. This method combines multiplexed high-fidelity PCR amplification with Oxford Nanopore sequencing and custom in-house Perl and Python scripts for streamlined data processing, including barcode-based demultiplexing, strand reorientation, alignment to a pseudo-genome, manual editing-site inspection, and splicing variant identification and comparison. Using this platform, we analyzed the ndhB and ndhD transcripts, two chloroplast NAD(P)H dehydrogenase genes with the highest number of known editing sites, in an inducible CRISPR interference (iCRISPRi) system targeting MORF2, a key RNA-editing factor. Our results revealed MORF2 dosage-dependent reductions in C-to-U editing efficiency, with significant defects observed in the strongly repressed P1-12 line. Moreover, we identified an accumulation of intron-retaining ndhB transcripts, specifically in Dex-treated iCRISPRi lines, indicating impaired chloroplast splicing functions upon MORF2 suppression. The platform achieves single-molecule resolution, robust reproducibility, and high read coverage across biological replicates at a fraction of the cost of lncRNA-seq, making it broadly accessible. This study establishes premium PCR sequencing as a versatile, scalable, and affordable tool for targeted post-transcriptional analysis in plant organelles and expands our understanding of MORF2′s role in chloroplast RNA maturation.

Microarray-based analysis reveals a novel role of the miRNA-613/SNAI2/CXCR4 axis in atrial fibrillation

2025-06-23

Juxiang Li , Y. Qi , Longlong Hu +1 more | PLoS ONE

Atrial fibrillation (AF) can lead to substantial morbidity and mortality in clinic. The previous studies demonstrated that miRNAs were closely associated with several cardiovascular diseases, however, the role of miRNAs … Atrial fibrillation (AF) can lead to substantial morbidity and mortality in clinic. The previous studies demonstrated that miRNAs were closely associated with several cardiovascular diseases, however, the role of miRNAs in the pathogenesis of AF has not been fully elucidated. In order to investigate the important role of miRNA in the mechanisms of AF, we conducted the study through bioinformatics analysis. We downloaded the miRNA expression profile (GSE68475) and mRNA expression profile (GSE31821) from the Gene Expression Omnibus (GEO) database to explore the differentially expressed miRNAs and mRNAs. The criteria for significant differentially expressed miRNA and mRNA using the R limma package were: adjusted P-value < 0.05, log2fold-change ≥ 1. The target mRNAs related to differentially expressed miRNAs of AF were predicted by using Functional enrichment analysis tool. We Screened overlapped mRNAs based on differentially expressed mRNAs and miRNA related mRNAs using Draw Venn Diagram. GO enrichment analysis and KEGG pathway analysis were conducted to explore the role of miRNAs and mRNAs in the pathogenesis of AF. A total of 70 differentially expressed miRNAs were screened including 33 up-regulated miRNAs and 34 downregulated miRNAs. All of 94 differentially expressed mRNAs were screened including 56 up-regulated mRNAs and 38 downregulated mRNAs. There were three co-expressed up-regulated differentially expressed genes, including CXCR4, SNAI2, and FHL1. We showed the results of GO functional enrichment analysis and KEGG pathway analysis ranked by enrichment score (-log P value) respectively. Compared with patients of normal sinus rhythm, miRNA-613 was significantly down-regulated in patients with AF. We demonstrated that SNAI2 and CXCR4 may target genes of miRNA-613 for the first time. Our findings may provide new ideas for clarifying the molecular mechanism of atrial fibrillation.

X-chromosome upregulation operates on a gene-by-gene basis at RNA and protein levels

2025-06-23

Ryan Allsop , Jeffrey Boeren , Beatrice Tan +7 more

Gene dosage compensation mechanisms are crucial for mammalian development. In mice, recent findings show that cells can sense the number of X chromosomes. Loss or inactivation of one of the … Gene dosage compensation mechanisms are crucial for mammalian development. In mice, recent findings show that cells can sense the number of X chromosomes. Loss or inactivation of one of the two X chromosomes is compensated by upregulating the remaining active X chromosome, a process termed X-chromosome upregulation (XCU). However, how cells sense X-chromosome dosage and induce XCU remains unclear. Here, we show that heterozygous X chromosome fragment deletions in mouse pluripotent stem cells induces XCU in trans, and that compensation takes place at the mRNA and protein level. Furthermore, we found that inducing gene silencing in cis on autosomes induces gene dosage compensation in trans. This work provides significant insights into the molecular foundations of mammalian gene dosage compensation.

ADAR1 Restricts Poliovirus Infection through dual disruption of translation initiation and protein coding

2025-06-23

Damian Kim , Frank McCarthy , Kathy Li +4 more | bioRxiv (Cold Spring Harbor Laboratory)

Viral RNAs (vRNAs) interact with hundreds of host proteins, but how these interactions shape viral infection remains largely unknown. Here we developed a mass-spectrometry approach to identify cellular RNA-binding proteins … Viral RNAs (vRNAs) interact with hundreds of host proteins, but how these interactions shape viral infection remains largely unknown. Here we developed a mass-spectrometry approach to identify cellular RNA-binding proteins (RBPs) that enhance or inhibit infection by modulating vRNA translation. We identified 130 RBPs that associate with polysomes in human cells infected with poliovirus, including known regulators of cap-independent translation from the viral internal ribosome entry site (IRES). We find that adenosine deaminase acting on RNA (ADAR1) is recruited to polysomes during infection and edits 3 sites in the vRNA, reducing viral replication. Two edits occur in the IRES and impair internal translation initiation, while the third edit occurs in the coding region and triggers an amino acid substitution. Incorporation of this threonine-to-alanine mutation into the viral genome attenuated replication. We conclude that ADAR1 restricts poliovirus infection by both reducing vRNA translation and introducing a recurrent coding error.

White Matter Disease in a Toddler With New‐Onset Seizures

2025-06-22

Olivia Viscuso , Bhairav Patel , James B. Gibson +1 more | Annals of the Child Neurology Society

Accurate interpretation of inosines in RNAs: recognized as G is more than basepairing with C

2025-06-21

Yuange Duan | Epigenomics

Adenosine-to-inosine (A-to-I) mRNA editing epigenetically mimics A-to-G mutations. While literatures usually state "inosines are recognized as guanosines," sometimes we also see expressions like "inosines basepair with cytosines." In this article, … Adenosine-to-inosine (A-to-I) mRNA editing epigenetically mimics A-to-G mutations. While literatures usually state "inosines are recognized as guanosines," sometimes we also see expressions like "inosines basepair with cytosines." In this article, I stress that the two expressions are not equivalent at all for the following reasons. (1) inosines are recognized as guanosines only when located in mRNAs, not tRNAs; (2) even when being recognized as guanosines, inosines can basepair with cytosine and uracil; (3) when inosine pairs with cytosine, the basepairing efficiency differs from G:C pairing. Therefore, we should be cautious when introducing A-to-I RNA editing. "inosines basepair with cytosines" is an inaccurate or incomplete interpretation and should be corrected as "inosines in mRNAs are recognized as guanosines."

RNA Adenosine Deaminases Modulate Oxidative Stress-Mediated Hepatocellular Carcinoma Cell Damage via KEAP1/Nrf2 Axis

2025-06-20

Changquan Li , Haitao Wang | Journal of Surgery & Anesthesia Research

This study investigates the role of RNA adenosine deaminases (ADAR1/2) in oxidative stress-induced Hepatocellular Carcinoma (HCC) cell damage. We demonstrate that hydrogen peroxide (H₂O₂)-mediated oxidative stress downregulates ADAR1/2 expression, impairing … This study investigates the role of RNA adenosine deaminases (ADAR1/2) in oxidative stress-induced Hepatocellular Carcinoma (HCC) cell damage. We demonstrate that hydrogen peroxide (H₂O₂)-mediated oxidative stress downregulates ADAR1/2 expression, impairing A-to-I RNA editing of antioxidant genes. Mechanistically, ADAR2 deficiency reduces KEAP1 editing, activating Nrf2 signaling and promoting HCC cell survival. These findings establish ADARs as critical regulators of redox homeostasis in HCC, providing novel therapeutic targets.

The BPIFA2 - LRP5 axis orchestrates mitochondrial dysfunction to mediate kidney injury in lupus nephritis

2025-06-20

Yunhe Liu , Yuexin Tian , Han Wang +7 more | International Immunopharmacology

Kidney injury, particularly the processes of inflammation and fibrosis, is a critical pathological feature of Lupus nephritis (LN). BPI fold-containing family A member 2 (BPIFA2), a recently identified parotid gland … Kidney injury, particularly the processes of inflammation and fibrosis, is a critical pathological feature of Lupus nephritis (LN). BPI fold-containing family A member 2 (BPIFA2), a recently identified parotid gland secretory protein, serves as an early biomarker for acute kidney injury (AKI) and has been shown to promote AKI development. However, the role of BPIFA2 in regulating LN remains largely unclear. In this study, we detected significant BPIFA2 expression in renal tubular epithelial cells of LN patients and animal models, which was strongly correlated with the degree of renal injury. Specifically, targeted knockdown of BPIFA2 in renal tubular epithelial cells significantly alleviated renal tubular injury in LN mice, while its overexpression exacerbated the injury. Mechanistically, under LN conditions, we found that BPIFA2 interacted with low density lipoprotein receptor-related protein 5 (LRP5) in HK-2 cells, resulting in mitochondrial dysfunction and ultimately leading to renal tubular injury. Collectively, our findings not only reveal a novel regulatory mechanism of renal tubular injury in LN but also suggest BPIFA2 as a promising therapeutic target for mitigating LN-associated kidney injury.

A Comprehensive Study of Circulating Blood Linear RNA nominates CD55 and DLD as novel causal genes and early-stage biomarkers for Parkinson Disease

2025-06-20

Aleksandra Beric , Sarp Sahin , Santiago Sánchez-Vicente +7 more | medRxiv (Cold Spring Harbor Laboratory)

ABSTRACT We leveraged transcriptomic data from 4,343 participants from four independent datasets to robustly identify and annotate circulating PD-associated transcripts. We identified 296 differentially expressed transcripts, 28 of which were … ABSTRACT We leveraged transcriptomic data from 4,343 participants from four independent datasets to robustly identify and annotate circulating PD-associated transcripts. We identified 296 differentially expressed transcripts, 28 of which were transcribed from known PD-associated loci. Further, we found a significant overlap between our findings and transcripts dysregulated in brain, as well as proteins differentially accumulated in CSF. Expression of the identified transcripts was affected by genetic background including ancestry and PD-related mutations, and nearly half of the identified transcripts were dysregulated before symptom onset. The differentially expressed transcripts were utilized to develop three predictive models that distinguished between PD and healthy controls with a ROC AUC of 0.727-0.733. The predictive models were capable of detecting PD transcriptomic signatures even before symptom onset. One transcript, DLD, showed particular promise as an early stage, minimally invasive PD biomarker that was differentially expressed in whole blood, brain and CSF. This transcript significantly related to PD in the eQTL analyses and in two of the three predictive models.

A Case Report and Review of the Literature on Hereditary Diffuse Leukoencephalopathy with Spheroids Presenting with Stroke-like Symptoms

2025-06-20

Jing Zhou , Yiming Wang , Yingte Wang +4 more | Clinical Neurology and Neurosurgery

Multi-omics machine learning classifier and blood transcriptomic signature of Parkinson’s disease

2025-06-20

Xianjun Dong , Ruifeng Hu , Ruoxuan Wang +7 more | Research Square (Research Square)

Early diagnosis and biomarker discovery to bolster the therapeutic pipeline for Parkinson's disease (PD) are urgently needed. In this study, we leverage the large-scale, whole-blood total RNA and DNA sequencing … Early diagnosis and biomarker discovery to bolster the therapeutic pipeline for Parkinson's disease (PD) are urgently needed. In this study, we leverage the large-scale, whole-blood total RNA and DNA sequencing data from the Accelerating Medicines Partnership in Parkinson's Disease (AMP PD) program to identify PD-associated RNAs, including both known genes and novel circular RNAs (circRNA) and enhancer RNAs (eRNAs). Initially, 874 known genes, 783 eRNAs, and 35 circRNAs were found differentially expressed in PD blood in the PPMI cohort (FDR < 0.05). Based on these findings, a novel multi-omics machine learning model was built to predict PD diagnosis with high performance (AUC = 0.89), which was superior to previous models. We further replicated this discovery in an independent PDBP/BioFIND cohort and confirmed 1,111 significant marker genes, including 491 known genes, 599 eRNAs, and 21 circRNAs. Functional enrichment analysis showed that the PD-associated genes are involved in neutrophil activation and degranulation, as well as the TNF-α signaling pathway. By comparing the PD-associated genes in blood with those in human brain dopamine neurons in our BRAINcode cohort, we found only 44 genes (9% of the known genes) showing significant changes with the same direction in both PD brain neurons and PD blood, among which are neuroinflammation-associated genes IKBIP, CXCR2, and NFKBIB. Our findings demonstrated consistently lower SNCA mRNA levels and the increased expression levels of VDR gene in the blood of early-stage PD patients. In summary, this study provides a generally useful computational framework for further biomarker development and early disease prediction. We also delineate a wide spectrum of the known and novel RNAs linked to PD that are detectable in circulating blood cells in a harmonized, large-scale dataset.

BiallelicSIDT2loss-of-function in a child with cerebellar ataxia and lysosomal dysfunction mimics impairment ofSIDT2in mice

2025-06-20

Tan Nguyen , Grace Yoon , Blake R. C. Smith +7 more | Journal of Medical Genetics

SIDT2 (Systemic Interference Deficient 1 Transmembrane Family Member 2) is a lysosomal membrane protein involved in RNA degradation via RNAutophagy. While animal models have indicated a link between SIDT2 deficiency … SIDT2 (Systemic Interference Deficient 1 Transmembrane Family Member 2) is a lysosomal membrane protein involved in RNA degradation via RNAutophagy. While animal models have indicated a link between SIDT2 deficiency and lysosomal storage disorders, no human cases have been reported. Here, we report a child with biallelic SIDT2 missense variants (p.Arg529Trp, p.Arg678Trp), who developed progressive neurological decline with cerebellar atrophy and Parkinsonian features. Functional studies revealed that the affected individual’s variants disrupted the ability of SIDT2 to interact with RNA. Fibroblasts from the affected individual showed impaired autophagy, characterised by abnormal accumulation of autophagy markers. In mouse models, Sidt2 was found to be highly expressed in the brain, particularly in the hippocampus and cerebellum. Sidt2 loss-of-function in mice resulted in not only impaired autophagy in the brain but also neurological dysfunction, including motor incoordination and eventual seizures. These findings suggest that SIDT2 deficiency contributes to both autophagic dysfunction and neurodegenerative processes, providing insight into a potential role in human neurological disease.

Oxidative Stress-Induced ADAR1/2 Dysfunction Promotes Hepatocellular Carcinoma Cell Damage via Redox Imbalance

2025-06-20

Changquan Li | Journal of Surgery & Anesthesia Research

Hepatocellular Carcinoma (HCC) progression is tightly linked to oxidative stress, yet the role of RNA Adenosine Deaminases (ADARs) in this process remains unclear. Here, we demonstrate that hydrogen peroxide (H₂O₂)-induced … Hepatocellular Carcinoma (HCC) progression is tightly linked to oxidative stress, yet the role of RNA Adenosine Deaminases (ADARs) in this process remains unclear. Here, we demonstrate that hydrogen peroxide (H₂O₂)-induced oxidative stress downregulates ADAR1/2 expression in HCC cells, leading to reduced A-to-I RNA editing and exacerbated cell damage. Mechanistically, ADAR loss impaired antioxidant defense by dysregulating SOD2/CAT expression and disrupting Nrf2 signaling. Our findings establish ADARs as critical regulators of redox homeostasis in HCC, offering novel therapeutic opportunities.

Tight Spaces, Tighter Signals: Spatial Constraints as Drivers of Peripheral Myelination

2025-06-18

Luca Bartesaghi , Basilio Giangreco , Vanessa Chiappini +6 more | Cells

Peripheral myelination is driven by the intricate interplay between Schwann cells and axons, coordinated through molecular signaling and the structural organization of their shared environment. While the biochemical regulation of … Peripheral myelination is driven by the intricate interplay between Schwann cells and axons, coordinated through molecular signaling and the structural organization of their shared environment. While the biochemical regulation of this process has been extensively studied, the influence of spatial architecture and mechanical cues remains poorly understood. Here, we use in vitro co-culture models-featuring microfluidic devices and hydrogel-based scaffolds-to explore how extracellular organization, cellular density, and spatial constraints shape Schwann cell behavior. Our results show that (i) pro-myelinating effects triggered by ascorbic acid administration is distally propagated along axons in Schwann cell-DRG co-cultures, (ii) ascorbic acid modulates Neuregulin-1 expression, (iii) a critical threshold of cellular density is required to support proper Schwann cell differentiation and myelin formation, and (iv) spatial confinement promotes myelination in the absence of ascorbic acid. Together, these findings highlight how spatial and structural parameters regulate the cellular and molecular events underlying peripheral myelination, offering new physiologically relevant models of myelination and opening new avenues for peripheral nerve repair strategies.

Ciprofloxacin disrupts testosterone synthesis in mice via downregulating StAR expression through NR4A1 pathway

2025-06-18

Lirui Hou , Yuhan Fu , Yue Zhao +5 more | Ecotoxicology and Environmental Safety

Progenitor Cell Dynamics in Androgenetic Alopecia: Insights from Spatially Resolved Transcriptomics

2025-06-17

Sasin Charoensuksira , Piyaporn Surinlert , Aungkana Krajarng +7 more | International Journal of Molecular Sciences

Androgenetic alopecia (AGA) is marked by the progressive miniaturization of hair follicles (HFs) and hair thinning, driven by a decline in the progenitor cells critical for hair regeneration. Despite this, … Androgenetic alopecia (AGA) is marked by the progressive miniaturization of hair follicles (HFs) and hair thinning, driven by a decline in the progenitor cells critical for hair regeneration. Despite this, the mechanisms responsible for progenitor cell depletion remain largely unclear. To investigate transcriptional alterations in the progenitor cell regions of AGA patients while maintaining the spatial tissue context, we employed the GeoMX Digital Spatial Profiling (DSP) platform, which enables a precise comparison with healthy controls. Our analysis revealed the significant upregulation of genes associated with extracellular matrix (ECM) organization and the epithelial-mesenchymal transition (EMT), including FN1, TWIST1, and TGFB2 in the progenitor cell region of the HFs. Correspondingly, protein expression data confirmed increased levels of the protein products of these genes in the affected areas, underscoring their roles in the disease's progression. These molecular changes suggest an environment conducive to the EMT, potentially contributing to the loss of progenitor cells and indicating a fibrogenic shift within the HF microenvironment. Additionally, our study highlights the influence of peri-infundibular immune cell infiltration on these molecular changes, suggesting that immune-mediated microinflammation may contribute to the fibrogenic environment and progenitor cell loss in the AGA. These findings demonstrate the utility of spatial transcriptomics in identifying potential therapeutic targets and advancing our understanding of AGA's molecular mechanisms, offering avenues for developing targeted treatment strategies.

Oral tofacitinib for generalized Dowling-Degos disease with refractory pruritus: a case report

2025-06-17

Yang Luan , Ming Jing , Zhenqiang Ruan +3 more | Journal of Dermatological Treatment

To evaluate the efficacy and safety of the Janus kinase (JAK) inhibitor tofacitinib in generalized Dowling-Degos disease (DDD) with refractory pruritus and to comparatively analyze its advantages and limitations versus … To evaluate the efficacy and safety of the Janus kinase (JAK) inhibitor tofacitinib in generalized Dowling-Degos disease (DDD) with refractory pruritus and to comparatively analyze its advantages and limitations versus existing therapies. We report a 61-year-old male with a 20-year history of generalized DDD presenting with pruritic erythematous-to-brown papules and macules refractory to conventional therapies. Multimodal diagnostic evaluations, including dermoscopy and histopathology confirmed the diagnosis, with pruritus severity quantified using the Numerical Rating Scale (NRS). The patient was initially treated with oral acitretin (20 mg/day) combined with prednisone (20 mg/day) for 1 month, followed by maintenance monotherapy with tofacitinib (5 mg twice daily). Oral acitretin exacerbated the pruritus. Following transition to tofacitinib, pruritus was improved from a baseline NRS score of 9 to 5-6 within 1 month. At four-month follow-up, pruritus scores further stabilized at 2-3, accompanied by lesion darkening without recurrence. No adverse events occurred. Tofacitinib represents a promising therapeutic option for DDD with refractory pruritus.

Rare Missense Variants in MYO7A and OTOP2 Genes in a South Korean Meniere Disease Cohort

2025-06-17

Mai T. Pham , Pablo Cruz‐Granados , Seung Hyun Jang +5 more | medRxiv (Cold Spring Harbor Laboratory)

ABSTRACT Meniere disease (MD) is a polygenic condition defined by episodes of vertigo associated with sensorineural hearing loss and tinnitus. Genetic studies in familial MD in East Asian population are … ABSTRACT Meniere disease (MD) is a polygenic condition defined by episodes of vertigo associated with sensorineural hearing loss and tinnitus. Genetic studies in familial MD in East Asian population are limited and the potential MD genes remain to be established in non-Finnish European populations. By exome sequencing and rare variant analysis, we search for existing and novel genes associated with MD in a South Korean cohort of 16 MD individuals with bilateral sensorineural hearing loss. We have found one individual with two rare missense variants in the OTOP2 gene, a new candidate gene for MD and three heterozygous variants in the MYO7A gene, supporting the hypothesis of biallelic inheritance. Protein modelling was conducted on three rare missense variants in OTOP2 to further elucidate functional consequences. The structural and functional implications inferred from these models suggest a likely pathogenic role, providing additional insights into the molecular mechanisms underlying MD.

IFN alpha inducible protein 27 (IFI27) acts as a positive regulator of PACT-dependent PKR activation after RNA virus infections

2025-06-16

Darío López-García , Vanessa Rivero , Laura Villamayor +1 more | PLoS Pathogens

Protein kinase R (PKR) expression is induced by interferons. This protein is activated by double-stranded (ds) RNAs or RNAs containing duplex regions, produced after different stimuli, such as after viral … Protein kinase R (PKR) expression is induced by interferons. This protein is activated by double-stranded (ds) RNAs or RNAs containing duplex regions, produced after different stimuli, such as after viral infections, leading to the phosphorylation of the eukaryotic translation initiation factor 2α (eIF2α), and subsequently inhibiting cellular and viral protein translation. This function may lead to different effects such as to impairing the replication of RNA viruses by inhibiting viral protein translation, and to modulating the innate immune responses after viral infections by affecting the translation of effector proteins. In this work, we identify, for the first time, an interaction of IFN alpha inducible protein 27 (IFI27) with PKR-activating protein (PACT or PRKRA) and with PKR, showing that the interaction of IFI27 with PACT is likely mediated by dsRNAs or RNAs containing duplex regions, and that the interaction of IFI27 with PKR is PACT-dependent. Interestingly, using IFI27 knocked-down, knocked-out and overexpressing tumour-derived, established cells, we show that these interactions trigger a potentiation of the activity of PKR and, therefore, a decrease in protein translation. Moreover, we find that IFI27 increases PKR function in cells infected with different RNA viruses such as Severe Acute Respiratory virus 2 (SARS-CoV-2), and Vesicular Stomatitis virus (VSV), and in cells transfected with the dsRNA analog poly(I:C), suggesting a broad effect of IFI27 on PKR activation. Moreover, we show that IFI27 expression increases the formation of stress granules (SGs) at the cell cytoplasm, correlating with the increased PKR activation mediated by IFI27, as it has been shown that the translational arrest induced by activated PKR leads to the formation of SGs. Mechanistically, we describe that this ability of IFI27 to activate PKR is dependent on its interaction with PACT. Further understanding of the regulation of PKR activity will allow us to develop new antiviral drugs to modulate this signalling axis, which is crucial in RNA virus infections.

A Fuzzy Logic-Based Computational Framework for Precision Triage in Androgenetic Alopecia: A Simulated Biomarker-Driven Approach

2025-06-16

Mohammed S. Al-Samarraay , Aws A. Magableh , Raja Azlina Raja Mahmood +7 more | International Journal of Computational Intelligence Systems

The Attenuated Phenotype of CNTNAP1‐Related Neuropathy Mimics Spastic–Dystonic Cerebral Palsy

2025-06-16

Magdalena Krygier , Michael Zech , Magdalena Chylińska +6 more | American Journal of Medical Genetics Part A

ABSTRACT CNTNAP1 encodes a contactin‐associated protein 1, which is essential for formation and organization of myelinated nerve fibers. Biallelic pathogenic variants in CNTNAP1 cause a severe congenital hypomyelinating neuropathy, characterized … ABSTRACT CNTNAP1 encodes a contactin‐associated protein 1, which is essential for formation and organization of myelinated nerve fibers. Biallelic pathogenic variants in CNTNAP1 cause a severe congenital hypomyelinating neuropathy, characterized by hypotonia, arthrogryposis, respiratory failure, and early lethality. We describe two brothers, seven and 13 years old, with spastic tetraparesis and limb dystonia, in whom we identified compound heterozygous variants in CNTNAP1 . Comprehensive neurophysiological evaluation revealed an unusual, asymmetric pattern of hypomyelination that spared lower limb nerves. Moreover, brain neuroimaging showed only mild terminal zone hypomyelination. This report extends the phenotypic spectrum of CNTNAP1 encephalopathy to primarily upper motor neuron disease with the predominant spastic features. In addition, it provides further evidence for the association of CNTNAP1 with dystonia. Importantly, CNTNAP1 mutations should be suspected in individuals with unexplained hypotonia and pyramidal syndrome even in the absence of apparent hypomyelination on brain imaging and normal conduction velocities in routinely examined nerves.

POLR3A rare variants in a patient with intellectual disability, ataxic gait and cortical malformations: a case-report

2025-06-15

C. Florio , Federica Mirabella , Adriana Prato +3 more | The Italian Journal of Pediatrics/Italian journal of pediatrics

Recessive mutations in POLR3A exhibit considerable phenotypic diversity, spanning from severe childhood-onset hypomyelinating leukodystrophic syndrome to less severe gait disorders, which may present later in life and may be accompanied … Recessive mutations in POLR3A exhibit considerable phenotypic diversity, spanning from severe childhood-onset hypomyelinating leukodystrophic syndrome to less severe gait disorders, which may present later in life and may be accompanied by additional non-neurological symptoms. In this study, we report a new case of rare POLR3A variants in a 6-year-old female patient sharing common genetic and neuropsychological profiles of POLR3-related disorders, although without revealing the classic MRI phenotype and severe clinical signs of POLR3-related leukodystrophy, such as diffuse hypomyelination. Our probe was born after full term pregnancy complicated by Intrauterine Growth Restriction and risk of preterm birth treated with tocolytics during the last weeks of pregnancy. On the second day of life, tremors in the lower and upper limbs were detected and lasted until the second month of life. At the age of 6 months, she was diagnosed with hypotonia. The child showed a delay in the stages of psychomotor development and a slowing of the language. Brain MRI performed at the age of 5 years revealed mild and symmetrical ectasia of the lateral ventricles, mild hypoplasia of the cerebellar vermis and brainstem with wide communication between the fourth ventricle and the cisterna magna. Neurological examination revealed dyslalia, mild generalized hypotonia, ataxic gait, motor coordination and balance deficits, while the Wechsler Intelligence Scale for Children revealed the presence of mild intellectual disability. A clinical exome and neurodevelopmental multigenic analysis revealed two variants of the POLR3A gene in compound heterozygosity (c.1795 C > A and c.1289 + 3 A > G) previously described in the literature and a novel and not yet reported CACNA2D2 variant (c.2929 C > T). Beside the shared genetic and neuropsychological findings, the distinctive MRI and classical clinical signs of POLR3-related leukodystrophy have not been revealed in our case. This finding underscores the need to expand the diagnostic approach for POLR3A-related disorders, highlighting the significance of differentiating subtle clinical signs and promoting the use of genetic testing, especially in younger patients who may not yet display the typical clinical and MRI patterns. Further studies are necessary to shed light on different pathogenic mechanisms potentially responsible for the heterogeneous phenotype associated with POLR3-related disorders.

IKZF1: An important target for the treatment of autoimmune diseases

2025-06-13

Siyuan Bu , Cong Ye , Shaozhe Cai +1 more | Clinical Immunology

The landscape of pediatric genetic white matter disorders at a tertiary referral hospital in Upper Egypt and the report of 31 novel variants

2025-06-13

Mahmoud M. Noureldeen , Maha S. Zaki , Karima Rafat +2 more | The Italian Journal of Pediatrics/Italian journal of pediatrics

Abstract Background Leukodystrophies (LDs) and genetic leukoencephalopathies (GLEs) encompass the spectrum of genetic white matter disorders (GWMDs). Despite their clinical significance, limited studies have investigated GWMDs in Egypt. Therefore, this … Abstract Background Leukodystrophies (LDs) and genetic leukoencephalopathies (GLEs) encompass the spectrum of genetic white matter disorders (GWMDs). Despite their clinical significance, limited studies have investigated GWMDs in Egypt. Therefore, this study aimed to characterize pediatric patients diagnosed with GWMDs in the Beni-Suef Governorate, Upper Egypt. Methods We reviewed the records of patients diagnosed with GWMDs who presented over five years to the pediatric neurology clinic of a tertiary care hospital in Beni-Suef Governorate, Upper Egypt. The study included 142 patients aged < 18 years diagnosed with GWMD confirmed by brain imaging, metabolic, and/or molecular genetic testing. Patients were classified as LDs or GLEs per the 2015 Global Leukodystrophy Initiative Consortium (GLIA) criteria. Results Fifty-six cases were identified to have LDs, while 86 were classified as GLEs. Metachromatic leukodystrophy (MLD) was the most common LD (13 patients), followed by megalencephalic leukoencephalopathy with subcortical cysts (MLC) (10 patients). The most common GLEs were lysosomal storage disorders (LSDs) (22 patients,) followed by Cockayne syndrome (11 patients), along with other miscellaneous disorders. The cumulative incidence of GWMDs in children under 18 was estimated at 10.8 cases per 100,000 population during the five-year study period. Thirty-one novel variants were identified, comprising 10 for LDs and 21 for GLEs. The mortality rate was 39.3% and 22.1% among patients with LDs and GLEs, respectively. Conclusions This study presents the first cohort of GWMDs reported from the Beni-Suef Governorate, Upper Egypt. The study provides significant data regarding regional etiological patterns, clinical trajectories, and molecular profiles. Additionally, the study findings provide a foundational framework for establishing a national GWMD registry and inform future diagnostic and therapeutic strategies.

Abstract P2-04-11: Selective Pre-clinical Targeting of CD44+ ADAR1+ Triple Negative Breast Cancer

2025-06-13

Wenxue Ma , Jessica Pham , Emma Klacking +7 more | Clinical Cancer Research

Abstract Background: Triple-negative breast cancer (TNBC) represents a challenging subtype in breast cancer with limited therapeutic options. Both CD44 and ADAR1 are biomarkers associated with tumor progression, metastasis, and therapeutic … Abstract Background: Triple-negative breast cancer (TNBC) represents a challenging subtype in breast cancer with limited therapeutic options. Both CD44 and ADAR1 are biomarkers associated with tumor progression, metastasis, and therapeutic resistance in TNBC. This study evaluates the efficacy of Rebecsinib, an experimental small-molecule anti-cancer drug designed to inhibit the splicing-mediated activation of the enzyme ADAR1 (adenosine deaminase acting on RNA 1), in targeting CD44+ and ADAR1+ cells in TNBC preclinical humanized mouse models. Methods: Triple-negative breast cancer MDA-MB-231 cell line-derived xenograft (CDX) models were established in Rag2-/-gc-/- and NSG-SGM3 mice. First, MDA-MB-231 cells were lentivirally transduced with an ADAR-nanoluciferase-GFP reporter. Following in vivo imaging (IVIS, Caliper) to detect ADAR1-nanoluciferase reporter activity, engrafted mice were treated with DMSO vehicle control, Rebecsinib at a dose of 10 mg/kg by intravenous (IV), or Rebecsinib at a dose of 15 mg/kg by oral (PO) gavage, twice a week for two weeks. Tumor burden was assessed using in vivo imaging system (IVIS) 200. Single-cell suspensions from peripheral blood, lung, liver, spleen, and bone marrow were analyzed by flow cytometry (FACS) to determine the percentages of CD44+ and ADAR1+ cells. Results: Both IV and oral administration of Rebecsinib significantly reduced CD44+ cells in peripheral blood (p &lt; 0.05), lung (p &lt; 0.01), liver (p &lt; 0.01), and spleen (p &lt; 0.05) in Rag2-/-gc-/- models (Student t test). A similar reduction in ADAR1+ cells was also observed in the lung (p &lt; 0.05), liver (p &lt; 0.01), and spleen (p &lt; 0.01) following Rebecsinib treatment in NSG-SGM3 models (Student t test). IVIS imaging revealed a substantial decrease in tumor signals in Rebecsinib-treated groups compared to the controls (p = 0.02, student t test). Combination therapy of Rebecsinib with Fedratinib demonstrated enhanced inhibition of tumor cell proliferation, suggesting a synergistic effect (p &lt; 0.05, student t test). Conclusion: Rebecsinib effectively targets CD44+ and ADAR1+ breast cancer cells in preclinical TNBC models, demonstrating potential as a therapeutic option. These findings support further investigation of Rebecsinib in combination with other agents for treating TNBC. Citation Format: Wenxue Ma, Jessica Pham, Emma Klacking, Inge van der Werf, Neha Katragadda, Jenna Sneifer, Peggy Wentworth, Kendale Wirtjes, Ethan Lam, Sheldon Morris, James La Clair, Michael Burkart, Catriona Jamieson. Selective Pre-clinical Targeting of CD44+ ADAR1+ Triple Negative Breast Cancer [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P2-04-11.

ASR gene family: a case of tandem-drive evolution

2025-06-13

Nicolle Louise Ferreira Barros , João Pedro Carmo Filgueiras , Thomaz Stumpf Trenz +3 more | Frontiers in Molecular Biosciences

Introduction ABA, Stress, and Ripening (ASR) proteins are characterized by the presence of the ABA/WDS domain and are involved in plant development processes and tolerance to abiotic and biotic stresses. … Introduction ABA, Stress, and Ripening (ASR) proteins are characterized by the presence of the ABA/WDS domain and are involved in plant development processes and tolerance to abiotic and biotic stresses. Despite their importance as transcription factors or molecular chaperones, a complete understanding of their biological roles is limited by a lack of information on their mechanisms of action, protein structure, and evolutionary relationships between family members. Our previous molecular dynamics simulation analysis of rice OsASR5 suggested that H 91 , R 92 , H 93 , and K 94 , are the main residues involved in the interaction with DNA, essential for the transcription factor activity of this protein. However, the presence and conservation of the DNA-binding domain among ASR family members remain unknown. Likewise, there is a lack of phylogenetic analyses evaluating the evolutionary history of ASR proteins across major taxonomic groups, outside just the Solanum species. Methods To address these gaps, we conducted a phylogenetic study and protein sequence analyses to gain insights into the evolution of ASR genes in plants. We performed a genome-wide identification of ASR genes via HMMER, using the ABA/WDS domain, in 163 Archaeplastida genomes. Results and discussion Our results reveal that the potential origin of the ASR gene occurred in the common ancestor of Streptophytes (Charophytes and Embryophytes). Moreover, our study identifies ASR genes in seedless plants. The evolutionary relationship between 465 ASR homologs, found in 76 species, was estimated through maximum likelihood analysis. The results reinforce the rapid and dynamic evolution of the ASR gene family, reflected by the low support in the deep nodes of the phylogeny and the great variation in the number of ASRs in the genomes evaluated, and in some cases their complete absence. As for diversification, tandem duplications seem to be the main mechanism involved. Regarding the conservation of residues in the domain, only two of the 78 are widely conserved, such as E 79 and H 93 . By analyzing the three-dimensional model, we noticed the interaction between them and we hypothesize that they are essential for the stabilization of the domain during interaction with DNA.

<scp>SASH1</scp> Mutations and Hereditary Disorders of Pigmentation: Review of Literature

2025-06-13

Anuradha Bishnoi , Aarushi Arunima , Keshavamurthy Vinay +2 more | Pigment Cell & Melanoma Research

ABSTRACT Dyschromatosis universalis hereditaria (DUH) is a rare genodermatosis characterized by asymptomatic hyper‐ and hypopigmented macules appearing in infancy and persisting for life. Although mutations in ABCB6 account for many … ABSTRACT Dyschromatosis universalis hereditaria (DUH) is a rare genodermatosis characterized by asymptomatic hyper‐ and hypopigmented macules appearing in infancy and persisting for life. Although mutations in ABCB6 account for many DUH cases, recently, the SAM and SH3 domain‐containing 1 (SASH1) gene has emerged as a key player in DUH. Additionally, SASH1 mutations have been associated with the pure‐lentiginous phenotype of familial pigmentation. In this review of literature, we found 22 different SASH1 mutations, most inherited in an autosomal dominant manner. These variants cause distinct phenotypes, including DUH, lentiginosis, and rarely, an autosomal recessive syndromic form with alopecia, palmoplantar keratoderma, and increased risk of malignancies. Functional studies have revealed that SASH1 acts as both a tumor suppressor and a pro‐melanogenic factor. It modulates key pathways such as p53–POMC–α‐MSH–MC1R–MITF and Gαs‐SASH1–IQGAP1–E‐cadherin pathways, affecting melanosome production, transport, and melanocyte migration. This unique dual role of SASH1 highlights its importance in melanocyte homeostasis and UV‐induced pigmentation. Understanding the role of SASH1 in regulating pigmentation can help foster novel therapeutic approaches for these genodermatoses and related pigmentary anomalies, ultimately improving patient care and outcomes.

Abstract P3-01-23: MELK controls stromal components through fibronectin modulation in highly aggressive breast cancers

2025-06-13

Mohd Mughees , Alex W. Tan , Jian Wang +6 more | Clinical Cancer Research

Abstract Background: Triple-negative breast cancer (TNBC) and inflammatory breast cancer (IBC) have limited therapeutic options due to advanced stage at diagnosis, high rate of metastasis, and lack of actionable targets. … Abstract Background: Triple-negative breast cancer (TNBC) and inflammatory breast cancer (IBC) have limited therapeutic options due to advanced stage at diagnosis, high rate of metastasis, and lack of actionable targets. In a previous study, we showed that MELK is a potential target in highly aggressive breast cancers; a driver of cell stemness, and promotes epithelial-mesenchymal transition and metastasis in a TNBC xenograft model. Fibronectin (FN1) produced by mesenchymal cells has been implicated in breast cancer metastasis, and previous studies have shown that FN1 expression is higher in TNBCs than in HER2-positive or hormone-receptor-positive breast cancers. Studies have also shown that an increase in FN1 can lead to the formation of extracellular fibronectin fibrils, which promote directional cancer cell motility. We previously showed that inhibition of MELK using siRNA against MELK and using a MELK inhibitor reduced the expression of FN1 in vitro and in a xenograft mouse model, suggesting that MELK kinase activity may be important for maintaining FN1 levels in TNBC. We hypothesized that MELK promotes the expression of FN1, which is involved in reorganization of the extracellular matrix. Methods and results: To determine if MELK promotes transition to mesenchymal-like stromal components, we used qPCR to evaluate the expression of FN1 in MDA-MB-231 clones with CRISPR-based MELK knockout. The clones C3 and C28 showed 0.54-fold ± 0.03-fold (p ≤ 0.01) and 0.76-fold ± 0.01-fold (p ≤ 0.001) reduction in expression of FN1 compared to Cas9 control cells. To determine if MELK promotes the motility and invasion of TNBC through FN1, we performed a migration assay by plating MELK-knockdown and MELK-inhibitor-treated TNBC/IBC cells in FN1-coated migration chambers. When MELK was knocked down using three siRNAs against MELK in MDA-MB-231 cells, the relative percentage of migration was reduced by 69.01% ± 0.03, 44.53% ± 0.05, and 11.80 ± 0.10, respectively, in the MELK-knockdown cells compared to the control cells transfected with non-targeting siRNA (p ≤ 0.001). Similarly, treatment of MDA-MB-231 and SUM149 cells with the MELK inhibitor 30e reduced the relative percentage migration by 30.45% ± 0.10 and 20.00% ± 0.14 compared to the respective DMSO-treated controls (p ≤ 0.05) for both comparisons. To evaluate the effect of MELK on stromal organization, we used the 3D Spheroid BME Invasion Assay to measure the invasion of MDA-MB-231 cells in the presence of FN1 and MELK-In-30e. The presence of FN1 enhanced the invasion of these cells compared to the control cells, and inhibition of MELK with MELK-In-30e reduced the invasion of these cells compared to the DMSO-treated control cells. To pave the path for clinical translation of MELK inhibitors, we evaluated three MELK inhibitors (MELK-In-17, MELK-In-30e, and OTS167) to find the inhibitor with the highest efficacy and lowest toxicity alone or in combination with paclitaxel in the murine TNBC 4T1 mouse model. All the treated groups exhibited a reduction in tumor growth compared to the vehicle-treated group. The MELK–In–30–treated group exhibited significant inhibition in tumor growth compared to the OTS167-treated group (p=0.002). The combinations of MELK inhibitors with paclitaxel did not show any synergism with respect to tumor growth inhibition. Conclusions and Future Directions: Our results showed that MELK promotes FN1 expression, which may be involved in stromal reorganization and contribute to tumor progression in TNBC. Currently, we are evaluating MELK inhibitors with other rational combination therapies in a triple-negative IBC xenograft model. Citation Format: Mohd Mughees, Alex Tan, Jian Wang, Savitri Krishnamurthy, Senthil Damodaran, Debu Tripathy, Wendy A. Woodward, Kevin Dalby, Chandra Bartholomeusz. MELK controls stromal components through fibronectin modulation in highly aggressive breast cancers [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P3-01-23.

1795-P: RIPK1 Mediates Nucleic Acid Sensor Expression and IFNγ+dsRNA-Induced β-Cell Death

2025-06-13

Renato Chaves Souto Branco , Christopher Contreras , Noyonika Mukherjee +5 more | Diabetes

Introduction and Objective: Type 1 diabetes (T1D) is characterized by immune-associated destruction of insulin-producing β-cells. Activation of antiviral response programs has been linked to β-cell demise in T1D, and previous … Introduction and Objective: Type 1 diabetes (T1D) is characterized by immune-associated destruction of insulin-producing β-cells. Activation of antiviral response programs has been linked to β-cell demise in T1D, and previous studies show that IFNγ + poly I:C (a synthetic double stranded RNA) synergistically elicit β-cell death. However, the mechanisms that underlie this form cytotoxicity are not fully understood. We previously identified receptor interacting protein kinase 1 (RIPK1) as a regulator of β-cell fate. Here, we hypothesized that RIPK1 promotes IFNγ + poly I:C-induced β-cell death. Methods: We treated NIT-1 β-cells with IFNγ + poly I:C for 24 hours and quantified cell death, caspase 3/7 activity, and gene expression. We tested control (CTL, non-targeting gRNA) and Ripk1 gene-edited (Ripk1Δ, gRNA targeting Ripk1 exons 2-3) NIT-1 β-cells and evaluated a small molecule RIPK1 kinase inhibitor (SZM679). Results: Treatment with IFNγ alone or poly I:C alone failed to increase caspase 3/7 activity or cell death in NIT-1 CTL cells, whereas co-treatment led to increases in both of these parameters. Blockade of RIPK1 with SZM679 or in NIT-1 Ripk1Δ cells protected from IFNγ + poly I:C-induced caspase 3/7 activation and cell death. RNAseq analysis revealed that RIPK1-deficient NIT-1 Ripk1Δ cells have decreased expression of nucleic acid sensors including Tlr3, cGas, Sting1, and Ifih1 compared to NIT-1 CTL cells, suggestive of a role for RIPK1 in nucleic acid sensing-related cell death. We also found that Ripk1 expression is increased 5-fold in 20- versus 8-week-old NOD mouse islets, and expression of the nucleic acid sensors Sting1, cGas, Zbp1, and Adar were also increased at this timepoint. Conclusion: Our data indicate that RIPK1 regulates nucleic acid sensor expression and mediates IFNγ + poly I:C-induced NIT-1 β-cell death. Studies are needed to determine the role of RIPK1 in nucleic acid sensor expression and β-cell demise in additional preclinical models of T1D. Disclosure R.C.S. Branco: None. C.J. Contreras: None. N. Mukherjee: None. E. Mather: None. L. Lin: None. K.A. Colglazier: Employee; Eli Lilly and Company. E.P. Cai: None. A.T. Templin: None. Funding U.S. Department of Veterans Affairs (IK2 BX004659 to ATT, I01 BX001060 to SEK); National Institutes of Health (P30 DK097512 to Indiana UniversityCenter for Diabetes and Metabolic Diseases, P30 DK017047 to University of Washington Diabetes Research Center); Ralph W. and Grace M. Showalter Research Trust (080657-00002B to ATT); VA Puget Sound Health Care System and the Richard L. Roudebush VA Medical Center.

Receptors from an ADAR trick

2025-06-09

Hayase Hakariya , Thorsten Stafforst | Nature Chemical Biology

Landscape of A-I RNA editing in mouse, pig, macaque, and human brains

2025-06-06

Conghui Li , Wei Lv , Zhiwei He +7 more | PubMed

Abstract Adenosine-to-inosine (A-I) RNA editing is one of the most abundant post-transcriptional RNA modification processes. However, the roles of A-I RNA editing in the evolution and functions of primate brains … Abstract Adenosine-to-inosine (A-I) RNA editing is one of the most abundant post-transcriptional RNA modification processes. However, the roles of A-I RNA editing in the evolution and functions of primate brains are underexplored. Here, we perform whole-genome and whole-transcriptome sequencing of 39 anatomically defined brain regions of adult Macaca fascicularis and identify 2 782 079 A-I editing sites, including 2009 recoding sites enriched in genes related to neurotransmission functions. Most of macaque brain A-I editing sites are detected in the cerebral cortex, cerebellum, and amygdala. The brain A-I editing activity is associated with the estimated proportion of neurons to some extent. Comparison of A-I editing in mouse, pig, macaque, and human brains reveals that primates exhibit higher editing levels, specifically enriched in genes encoding neurotransmitter receptors. We identify 478 598 conserved brain editing sites between human and macaque brains, mostly found in the cerebral cortex and enriched in genes related to the cytoskeletal system and ubiquitin-dependent protein degradation pathway. Our study sheds light on the importance of post-transcriptional A-I RNA editing in the evolution and function of nervous systems.