meCLICK-Seq, a Substrate-Hijacking and RNA Degradation Strategy for the Study of RNA Methylation.
ACS central science
The fates of RNA species in a cell are controlled by ribonucleases, which degrade them by exploiting the universal structural 2'-OH group. This phenomenon plays a key role in numerous transformative technologies, for example, RNA interference and CRISPR/Cas13-based RNA editing systems. These approaches, however, are genetic or oligomer-based and so have inherent limitations. This has led to interest in the development of small molecules capable of degrading nucleic acids in a targeted manner. Here we describe click-degraders, small molecules that can be covalently attached to RNA species through click-chemistry and can degrade them, that are akin to ribonucleases. By using these molecules, we have developed the meCLICK-Seq (methylation CLICK-degradation Sequencing) a method to identify RNA modification substrates with high resolution at intronic and intergenic regions. The method hijacks RNA methyltransferase activity to introduce an alkyne, instead of a methyl, moiety on RNA. Subsequent copper(I)-catalyzed azide-alkyne cycloaddition reaction with the click-degrader leads to RNA cleavage and degradation exploiting a mechanism used by endogenous ribonucleases. Focusing on N-methyladenosine (mA), meCLICK-Seq identifies methylated transcripts, determines RNA methylase specificity, and reliably maps modification sites in intronic and intergenic regions. Importantly, we show that METTL16 deposits mA to intronic polyadenylation (IPA) sites, which suggests a potential role for METTL16 in IPA and, in turn, splicing. Unlike other methods, the readout of meCLICK-Seq is depletion, not enrichment, of modified RNA species, which allows a comprehensive and dynamic study of RNA modifications throughout the transcriptome, including regions of low abundance. The click-degraders are highly modular and so may be exploited to study any RNA modification and design new technologies that rely on RNA degradation.
10.1021/acscentsci.0c01094
FTO-dependent demethylation of N6-methyladenosine regulates mRNA splicing and is required for adipogenesis.
Zhao Xu,Yang Ying,Sun Bao-Fa,Shi Yue,Yang Xin,Xiao Wen,Hao Ya-Juan,Ping Xiao-Li,Chen Yu-Sheng,Wang Wen-Jia,Jin Kang-Xuan,Wang Xing,Huang Chun-Min,Fu Yu,Ge Xiao-Meng,Song Shu-Hui,Jeong Hyun Seok,Yanagisawa Hiroyuki,Niu Yamei,Jia Gui-Fang,Wu Wei,Tong Wei-Min,Okamoto Akimitsu,He Chuan,Rendtlew Danielsen Jannie M,Wang Xiu-Jie,Yang Yun-Gui
Cell research
The role of Fat Mass and Obesity-associated protein (FTO) and its substrate N6-methyladenosine (m6A) in mRNA processing and adipogenesis remains largely unknown. We show that FTO expression and m6A levels are inversely correlated during adipogenesis. FTO depletion blocks differentiation and only catalytically active FTO restores adipogenesis. Transcriptome analyses in combination with m6A-seq revealed that gene expression and mRNA splicing of grouped genes are regulated by FTO. M6A is enriched in exonic regions flanking 5'- and 3'-splice sites, spatially overlapping with mRNA splicing regulatory serine/arginine-rich (SR) protein exonic splicing enhancer binding regions. Enhanced levels of m6A in response to FTO depletion promotes the RNA binding ability of SRSF2 protein, leading to increased inclusion of target exons. FTO controls exonic splicing of adipogenic regulatory factor RUNX1T1 by regulating m6A levels around splice sites and thereby modulates differentiation. These findings provide compelling evidence that FTO-dependent m6A demethylation functions as a novel regulatory mechanism of RNA processing and plays a critical role in the regulation of adipogenesis.
10.1038/cr.2014.151
PRMT5 methylome profiling uncovers a direct link to splicing regulation in acute myeloid leukemia.
Nature structural & molecular biology
Protein arginine methyltransferase 5 (PRMT5) has emerged as a promising cancer drug target, and three PRMT5 inhibitors are currently in clinical trials for multiple malignancies. In this study, we investigated the role of PRMT5 in human acute myeloid leukemia (AML). Using an enzymatic dead version of PRMT5 and a PRMT5-specific inhibitor, we demonstrated the requirement of the catalytic activity of PRMT5 for the survival of AML cells. We then identified PRMT5 substrates using multiplexed quantitative proteomics and investigated their role in the survival of AML cells. We found that the function of the splicing regulator SRSF1 relies on its methylation by PRMT5 and that loss of PRMT5 leads to changes in alternative splicing of multiple essential genes. Our study proposes a mechanism for the requirement of PRMT5 for leukemia cell survival and provides potential biomarkers for the treatment response to PRMT5 inhibitors.
10.1038/s41594-019-0313-z
Nuclear TARBP2 Drives Oncogenic Dysregulation of RNA Splicing and Decay.
Fish Lisa,Navickas Albertas,Culbertson Bruce,Xu Yichen,Nguyen Hoang C B,Zhang Steven,Hochman Myles,Okimoto Ross,Dill Brian D,Molina Henrik,Najafabadi Hamed S,Alarcón Claudio,Ruggero Davide,Goodarzi Hani
Molecular cell
Post-transcriptional regulation of RNA stability is a key step in gene expression control. We describe a regulatory program, mediated by the RNA binding protein TARBP2, that controls RNA stability in the nucleus. TARBP2 binding to pre-mRNAs results in increased intron retention, subsequently leading to targeted degradation of TARBP2-bound transcripts. This is mediated by TARBP2 recruitment of the mA RNA methylation machinery to its target transcripts, where deposition of mA marks influences the recruitment of splicing regulators, inhibiting efficient splicing. Interactions between TARBP2 and the nucleoprotein TPR then promote degradation of these TARBP2-bound transcripts by the nuclear exosome. Additionally, analysis of clinical gene expression datasets revealed a functional role for TARBP2 in lung cancer. Using xenograft mouse models, we find that TARBP2 affects tumor growth in the lung and that this is dependent on TARBP2-mediated destabilization of ABCA3 and FOXN3. Finally, we establish ZNF143 as an upstream regulator of TARBP2 expression.
10.1016/j.molcel.2019.06.001
Therapeutic Targeting of RNA Splicing Catalysis through Inhibition of Protein Arginine Methylation.
Fong Jia Yi,Pignata Luca,Goy Pierre-Alexis,Kawabata Kimihito Cojin,Lee Stanley Chun-Wei,Koh Cheryl M,Musiani Daniele,Massignani Enrico,Kotini Andriana G,Penson Alex,Wun Cheng Mun,Shen Yudao,Schwarz Megan,Low Diana Hp,Rialdi Alexander,Ki Michelle,Wollmann Heike,Mzoughi Slim,Gay Florence,Thompson Christine,Hart Timothy,Barbash Olena,Luciani Genna M,Szewczyk Magdalena M,Wouters Bas J,Delwel Ruud,Papapetrou Eirini P,Barsyte-Lovejoy Dalia,Arrowsmith Cheryl H,Minden Mark D,Jin Jian,Melnick Ari,Bonaldi Tiziana,Abdel-Wahab Omar,Guccione Ernesto
Cancer cell
Cancer-associated mutations in genes encoding RNA splicing factors (SFs) commonly occur in leukemias, as well as in a variety of solid tumors, and confer dependence on wild-type splicing. These observations have led to clinical efforts to directly inhibit the spliceosome in patients with refractory leukemias. Here, we identify that inhibiting symmetric or asymmetric dimethylation of arginine, mediated by PRMT5 and type I protein arginine methyltransferases (PRMTs), respectively, reduces splicing fidelity and results in preferential killing of SF-mutant leukemias over wild-type counterparts. These data identify genetic subsets of cancer most likely to respond to PRMT inhibition, synergistic effects of combined PRMT5 and type I PRMT inhibition, and a mechanistic basis for the therapeutic efficacy of PRMT inhibition in cancer.
10.1016/j.ccell.2019.07.003
HNRNPA2B1 Is a Mediator of m(6)A-Dependent Nuclear RNA Processing Events.
Alarcón Claudio R,Goodarzi Hani,Lee Hyeseung,Liu Xuhang,Tavazoie Saeed,Tavazoie Sohail F
Cell
N(6)-methyladenosine (m(6)A) is the most abundant internal modification of messenger RNA. While the presence of m(6)A on transcripts can impact nuclear RNA fates, a reader of this mark that mediates processing of nuclear transcripts has not been identified. We find that the RNA-binding protein HNRNPA2B1 binds m(6)A-bearing RNAs in vivo and in vitro and its biochemical footprint matches the m(6)A consensus motif. HNRNPA2B1 directly binds a set of nuclear transcripts and elicits similar alternative splicing effects as the m(6)A writer METTL3. Moreover, HNRNPA2B1 binds to m(6)A marks in a subset of primary miRNA transcripts, interacts with the microRNA Microprocessor complex protein DGCR8, and promotes primary miRNA processing. Also, HNRNPA2B1 loss and METTL3 depletion cause similar processing defects for these pri-miRNA precursors. We propose HNRNPA2B1 to be a nuclear reader of the m(6)A mark and to mediate, in part, this mark's effects on primary microRNA processing and alternative splicing. PAPERCLIP.
10.1016/j.cell.2015.08.011
Control of alternative splicing through siRNA-mediated transcriptional gene silencing.
Alló Mariano,Buggiano Valeria,Fededa Juan P,Petrillo Ezequiel,Schor Ignacio,de la Mata Manuel,Agirre Eneritz,Plass Mireya,Eyras Eduardo,Elela Sherif Abou,Klinck Roscoe,Chabot Benoit,Kornblihtt Alberto R
Nature structural & molecular biology
When targeting promoter regions, small interfering RNAs (siRNAs) trigger a previously proposed pathway known as transcriptional gene silencing by promoting heterochromatin formation. Here we show that siRNAs targeting intronic or exonic sequences close to an alternative exon regulate the splicing of that exon. The effect occurred in hepatoma and HeLa cells with siRNA antisense strands designed to enter the silencing pathway, suggesting hybridization with nascent pre-mRNA. Unexpectedly, in HeLa cells the sense strands were also effective, suggesting that an endogenous antisense transcript, detectable in HeLa but not in hepatoma cells, acts as a target. The effect depends on Argonaute-1 and is counterbalanced by factors favoring chromatin opening or transcriptional elongation. The increase in heterochromatin marks (dimethylation at Lys9 and trimethylation at Lys27 of histone H3) at the target site, the need for the heterochromatin-associated protein HP1alpha and the reduction in RNA polymerase II processivity suggest a mechanism involving the kinetic coupling of transcription and alternative splicing.
10.1038/nsmb.1620
Coordinated alterations in RNA splicing and epigenetic regulation drive leukaemogenesis.
Nature
Transcription and pre-mRNA splicing are key steps in the control of gene expression and mutations in genes regulating each of these processes are common in leukaemia. Despite the frequent overlap of mutations affecting epigenetic regulation and splicing in leukaemia, how these processes influence one another to promote leukaemogenesis is not understood and, to our knowledge, there is no functional evidence that mutations in RNA splicing factors initiate leukaemia. Here, through analyses of transcriptomes from 982 patients with acute myeloid leukaemia, we identified frequent overlap of mutations in IDH2 and SRSF2 that together promote leukaemogenesis through coordinated effects on the epigenome and RNA splicing. Whereas mutations in either IDH2 or SRSF2 imparted distinct splicing changes, co-expression of mutant IDH2 altered the splicing effects of mutant SRSF2 and resulted in more profound splicing changes than either mutation alone. Consistent with this, co-expression of mutant IDH2 and SRSF2 resulted in lethal myelodysplasia with proliferative features in vivo and enhanced self-renewal in a manner not observed with either mutation alone. IDH2 and SRSF2 double-mutant cells exhibited aberrant splicing and reduced expression of INTS3, a member of the integrator complex, concordant with increased stalling of RNA polymerase II (RNAPII). Aberrant INTS3 splicing contributed to leukaemogenesis in concert with mutant IDH2 and was dependent on mutant SRSF2 binding to cis elements in INTS3 mRNA and increased DNA methylation of INTS3. These data identify a pathogenic crosstalk between altered epigenetic state and splicing in a subset of leukaemias, provide functional evidence that mutations in splicing factors drive myeloid malignancy development, and identify spliceosomal changes as a mediator of IDH2-mutant leukaemogenesis.
10.1038/s41586-019-1618-0
CTCF-promoted RNA polymerase II pausing links DNA methylation to splicing.
Shukla Sanjeev,Kavak Ersen,Gregory Melissa,Imashimizu Masahiko,Shutinoski Bojan,Kashlev Mikhail,Oberdoerffer Philipp,Sandberg Rickard,Oberdoerffer Shalini
Nature
Alternative splicing of pre-messenger RNA is a key feature of transcriptome expansion in eukaryotic cells, yet its regulation is poorly understood. Spliceosome assembly occurs co-transcriptionally, raising the possibility that DNA structure may directly influence alternative splicing. Supporting such an association, recent reports have identified distinct histone methylation patterns, elevated nucleosome occupancy and enriched DNA methylation at exons relative to introns. Moreover, the rate of transcription elongation has been linked to alternative splicing. Here we provide the first evidence that a DNA-binding protein, CCCTC-binding factor (CTCF), can promote inclusion of weak upstream exons by mediating local RNA polymerase II pausing both in a mammalian model system for alternative splicing, CD45, and genome-wide. We further show that CTCF binding to CD45 exon 5 is inhibited by DNA methylation, leading to reciprocal effects on exon 5 inclusion. These findings provide a mechanistic basis for developmental regulation of splicing outcome through heritable epigenetic marks.
10.1038/nature10442
A methyl transferase links the circadian clock to the regulation of alternative splicing.
Sanchez Sabrina E,Petrillo Ezequiel,Beckwith Esteban J,Zhang Xu,Rugnone Matias L,Hernando C Esteban,Cuevas Juan C,Godoy Herz Micaela A,Depetris-Chauvin Ana,Simpson Craig G,Brown John W S,Cerdán Pablo D,Borevitz Justin O,Mas Paloma,Ceriani M Fernanda,Kornblihtt Alberto R,Yanovsky Marcelo J
Nature
Circadian rhythms allow organisms to time biological processes to the most appropriate phases of the day-night cycle. Post-transcriptional regulation is emerging as an important component of circadian networks, but the molecular mechanisms linking the circadian clock to the control of RNA processing are largely unknown. Here we show that PROTEIN ARGININE METHYL TRANSFERASE 5 (PRMT5), which transfers methyl groups to arginine residues present in histones and Sm spliceosomal proteins, links the circadian clock to the control of alternative splicing in plants. Mutations in PRMT5 impair several circadian rhythms in Arabidopsis thaliana and this phenotype is caused, at least in part, by a strong alteration in alternative splicing of the core-clock gene PSEUDO RESPONSE REGULATOR 9 (PRR9). Furthermore, genome-wide studies show that PRMT5 contributes to the regulation of many pre-messenger-RNA splicing events, probably by modulating 5'-splice-site recognition. PRMT5 expression shows daily and circadian oscillations, and this contributes to the mediation of the circadian regulation of expression and alternative splicing of a subset of genes. Circadian rhythms in locomotor activity are also disrupted in dart5-1, a mutant affected in the Drosophila melanogaster PRMT5 homologue, and this is associated with alterations in splicing of the core-clock gene period and several clock-associated genes. Our results demonstrate a key role for PRMT5 in the regulation of alternative splicing and indicate that the interplay between the circadian clock and the regulation of alternative splicing by PRMT5 constitutes a common mechanism that helps organisms to synchronize physiological processes with daily changes in environmental conditions.
10.1038/nature09470
FTO controls reversible mAm RNA methylation during snRNA biogenesis.
Mauer Jan,Sindelar Miriam,Despic Vladimir,Guez Théo,Hawley Ben R,Vasseur Jean-Jacques,Rentmeister Andrea,Gross Steven S,Pellizzoni Livio,Debart Françoise,Goodarzi Hani,Jaffrey Samie R
Nature chemical biology
Small nuclear RNAs (snRNAs) are core spliceosome components and mediate pre-mRNA splicing. Here we show that snRNAs contain a regulated and reversible nucleotide modification causing them to exist as two different methyl isoforms, m and m, reflecting the methylation state of the adenosine adjacent to the snRNA cap. We find that snRNA biogenesis involves the formation of an initial m isoform with a single-methylated adenosine (2'-O-methyladenosine, Am), which is then converted to a dimethylated m isoform (N,2'-O-dimethyladenosine, mAm). The relative m and m isoform levels are determined by the RNA demethylase FTO, which selectively demethylates the m isoform. We show FTO is inhibited by the oncometabolite D-2-hydroxyglutarate, resulting in increased m-snRNA levels. Furthermore, cells that exhibit high m-snRNA levels show altered patterns of alternative splicing. Together, these data reveal that FTO controls a previously unknown central step of snRNA processing involving reversible methylation, and suggest that epitranscriptomic information in snRNA may influence mRNA splicing.
10.1038/s41589-019-0231-8
Intragenic DNA methylation modulates alternative splicing by recruiting MeCP2 to promote exon recognition.
Maunakea Alika K,Chepelev Iouri,Cui Kairong,Zhao Keji
Cell research
Although the function of DNA methylation in gene promoter regions is well established in transcriptional repression, the function of the evolutionarily conserved widespread distribution of DNA methylation in gene body regions remains incompletely understood. Here, we show that DNA methylation is enriched in included alternatively spliced exons (ASEs), and that inhibition of DNA methylation results in aberrant splicing of ASEs. The methyl-CpG-binding protein MeCP2 is enriched in included ASEs, particularly those that are also highly methylated, and inhibition of DNA methylation disrupts specific targeting of MeCP2 to exons. Interestingly, ablation of MeCP2 results in increased histone acetylation and aberrant ASE-skipping events. We further show that inhibition of histone deacetylase (HDAC) activity leads to exon skipping that shows a highly significant degree of overlap with that caused by MeCP2 knockdown. Together, our data indicate that intragenic DNA methylation operates in exon definition to modulate alternative RNA splicing and can enhance exon recognition via recruitment of the multifunctional protein MeCP2, which thereby maintains local histone hypoacetylation through the subsequent recruitment of HDACs.
10.1038/cr.2013.110
Integrative analysis reveals functional and regulatory roles of H3K79me2 in mediating alternative splicing.
Li Tianbao,Liu Qi,Garza Nick,Kornblau Steven,Jin Victor X
Genome medicine
BACKGROUND:Accumulating evidence suggests alternative splicing (AS) is a co-transcriptional splicing process not only controlled by RNA-binding splicing factors, but also mediated by epigenetic regulators, such as chromatin structure, nucleosome density, and histone modification. Aberrant AS plays an important role in regulating various diseases, including cancers. METHODS:In this study, we integrated AS events derived from RNA-seq with H3K79me2 ChIP-seq data across 34 different normal and cancer cell types and found the higher enrichment of H3K79me2 in two AS types, skipping exon (SE) and alternative 3' splice site (A3SS). RESULTS:Interestingly, by applying self-organizing map (SOM) clustering, we unveiled two clusters mainly comprised of blood cancer cell types with a strong correlation between H3K79me2 and SE. Remarkably, the expression of transcripts associated with SE was not significantly different from that of those not associated with SE, indicating the involvement of H3K79me2 in splicing has little impact on full mRNA transcription. We further showed that the deletion of DOT1L1, the sole H3K79 methyltransferase, impeded leukemia cell proliferation as well as switched exon skipping to the inclusion isoform in two MLL-rearranged acute myeloid leukemia cell lines. Our data demonstrate H3K79me2 was involved in mediating SE processing, which might in turn influence transformation and disease progression in leukemias. CONCLUSIONS:Collectively, our work for the first time reveals that H3K79me2 plays functional and regulatory roles through a co-transcriptional splicing mechanism.
10.1186/s13073-018-0538-1
Regulation of alternative splicing by local histone modifications: potential roles for RNA-guided mechanisms.
Zhou Hua-Lin,Luo Guangbin,Wise Jo Ann,Lou Hua
Nucleic acids research
The molecular mechanisms through which alternative splicing and histone modifications regulate gene expression are now understood in considerable detail. Here, we discuss recent studies that connect these two previously separate avenues of investigation, beginning with the unexpected discoveries that nucleosomes are preferentially positioned over exons and DNA methylation and certain histone modifications also show exonic enrichment. These findings have profound implications linking chromatin structure, histone modification and splicing regulation. Complementary single gene studies provided insight into the mechanisms through which DNA methylation and histones modifications modulate alternative splicing patterns. Here, we review an emerging theme resulting from these studies: RNA-guided mechanisms integrating chromatin modification and splicing. Several groundbreaking papers reported that small noncoding RNAs affect alternative exon usage by targeting histone methyltransferase complexes to form localized facultative heterochromatin. More recent studies provided evidence that pre-messenger RNA itself can serve as a guide to enable precise alternative splicing regulation via local recruitment of histone-modifying enzymes, and emerging evidence points to a similar role for long noncoding RNAs. An exciting challenge for the future is to understand the impact of local modulation of transcription elongation rates on the dynamic interplay between histone modifications, alternative splicing and other processes occurring on chromatin.
10.1093/nar/gkt875
Interactions of gene expression, alternative splicing, and DNA methylation in determining nodule identity.
The Plant journal : for cell and molecular biology
Soybean nodulation is a highly controlled process that involves complex gene regulation at both transcriptional and post-transcriptional levels. In the present study, we profiled gene expression changes, alternative splicing events, and DNA methylation patterns during nodule formation, development, and senescence. The transcriptome data uncovered key transcription patterns of nodule development that included 9669 core genes and 7302 stage-specific genes. Alternative splicing analysis uncovered a total of 2323 genes that undergo alternative splicing events in at least one nodule developmental stage, with activation of exon skipping and repression of intron retention being the most common splicing events in nodules compared to roots. Approximately 40% of the differentially spliced genes were also differentially expressed at the same nodule developmental stage, implying a substantial association between gene expression and alternative splicing. Genome-wide-DNA methylation analysis revealed dynamic changes in nodule methylomes that were specific to each nodule stage, occurred in a sequence-specific manner, and impacted the expression of 1864 genes. An attractive hypothesis raised by our data is that increased DNA methylation may contribute to the efficiency of alternative splicing. Together, our results provide intriguing insights into the associations between gene expression, alternative splicing, and DNA methylation that may shape transcriptome complexity and proteome specificity in developing soybean nodules.
10.1111/tpj.14861
The importance of DNA methylation of exons on alternative splicing.
RNA (New York, N.Y.)
Alternative splicing (AS) contributes to proteome diversity. As splicing occurs cotranscriptionally, epigenetic determinants such as DNA methylation likely play a part in regulation of AS. Previously, we have shown that DNA methylation marks exons and that a loss of DNA methylation alters splicing patterns in a genome-wide manner. To investigate the influence of DNA methylation on splicing of individual genes, we developed a method to manipulate DNA methylation in vivo in a site-specific manner using the deactivated endonuclease Cas9 fused to enzymes that methylate or demethylate DNA. We used this system to directly change the DNA methylation pattern of selected exons and introns. We demonstrated that changes in the methylation pattern of alternatively spliced exons, but not constitutively spliced exons or introns, altered inclusion levels. This is the first direct demonstration that DNA methylation of exon-encoding regions is directly involved in regulation of AS.
10.1261/rna.064865.117