Abstract 3482: METTL3-mediated m6A modification controls splicing factor abundance and contributes to CLL progression
Yiming WuMeiling JinMike M. FernandezKevyn L. HartAijun LiaoStacey M. FernandesTinisha McDonaldZhenhua ChenDaniel RöthLucy GhodaGuido MarcucciMarkus KalkumRaju PillaiAlexey V. DanilovJianjun ChenJennifer R. BrownSteven T. RosenTanya SiddiqiLili Wang
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Abstract RNA splicing dysregulation is a hallmark of cancers and underlies the onset and progression of diseases. Chronic lymphocytic leukemia (CLL) is one of the most common adult leukemia in western countries. Spliceosome mutations occur in ~20% of patients. However, the mechanism for splicing defects in spliceosome unmutated CLL cases remains elusive. Through an integrative transcriptomic and proteomic analysis of primary CLL samples, we discovered proteins involved in RNA splicing are post-transcriptionally upregulated. Coupled with clinical annotation, we found spliceosome protein abundance is an independent risk factor and associated with poor prognosis. Splice variants found in CLL are highly overlapped with those driven by high spliceosome abundance but not splicing factor mutations, indicating high spliceosome abundance contributes to genetic lesion-independent splicing defects. To identify potential regulators for spliceosome, we proteome-widely analyzed the proteins that highly correlated with splicing factors expression. Analysis of 113 CLL samples has consistently identified METTL3 upregulation with positive correlation with 77.6% of detected splicing factors. METTL3 is an RNA methyltransferase that modifies N6-methyladenosine (m6A) on mRNA and regulates the translation of m6A-installed transcripts. m6A level on mRNA is increased in CLL cells with differential m6A highly enriched on splicing related transcripts. Moreover, high METTL3 expression in CLL is also associated with poor clinical outcomes. These results suggested that METTL3 translationally controls splicing factors through m6A and plays a role in CLL progression. Toward this end, we demonstrated that METTL3 is essential for CLL growth in both in vitro and in vivo studies. Knock out (KO) and pharmaceutical inhibition of METTL3 decreased cell growth and splicing factor expression. Overexpression of wildtype but not catalytic mutant METTL3 restored both defects in METTL3 KO cells, indicating that the regulation of splicing factor is m6A-dependent. To dissect the underlying mechanism, we performed an integrated Ribo-seq, RNA-seq, and MeRIP-seq on CLL cells with or without METTL3. KO of METTL3 decreased overall translation efficiency (TE) with RNA splicing as the most significantly affected pathway. Splicing factors with reduced TE displayed either hypo-m6A at stop codon region or hyper-m6A at CDS regions upon METTL3 KO, as direct or indirect targets of METTL3. Moreover, we found that m6A at stop codon and CDS regions regulates splicing factor translation via ribosome recycling and ribosome pausing, respectively. Taken together, our results uncovered a novel regulatory axis for METTL3 that controls splicing factor translation and contributes to CLL progression. Our study highlights a post-transcriptional layer of m6A modification as a major contributor to genetic lesion-independent splicing defects in CLL. Citation Format: Yiming Wu, Meiling Jin, Mike Fernandez, Kevyn Hart, Aijun Liao, Stacey M. Fernandes, Tinisha McDonald, Zhenhua Chen, Daniel Röth, Lucy Ghoda, Guido Marcucci, Markus Kalkum, Raju K. Pillai, Alexey V. Danilov, Jianjun Chen, Jennifer R. Brown, Steven T. Rosen, Tanya Siddiqi, Lili Wang. METTL3-mediated m6A modification controls splicing factor abundance and contributes to CLL progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3482.Keywords:
Spliceosome
Splicing factor
Exonic splicing enhancer
Abstract RNA splicing dysregulation is a hallmark of cancers and underlies the onset and progression of diseases. Chronic lymphocytic leukemia (CLL) is one of the most common adult leukemia in western countries. Spliceosome mutations occur in ~20% of patients. However, the mechanism for splicing defects in spliceosome unmutated CLL cases remains elusive. Through an integrative transcriptomic and proteomic analysis of primary CLL samples, we discovered proteins involved in RNA splicing are post-transcriptionally upregulated. Coupled with clinical annotation, we found spliceosome protein abundance is an independent risk factor and associated with poor prognosis. Splice variants found in CLL are highly overlapped with those driven by high spliceosome abundance but not splicing factor mutations, indicating high spliceosome abundance contributes to genetic lesion-independent splicing defects. To identify potential regulators for spliceosome, we proteome-widely analyzed the proteins that highly correlated with splicing factors expression. Analysis of 113 CLL samples has consistently identified METTL3 upregulation with positive correlation with 77.6% of detected splicing factors. METTL3 is an RNA methyltransferase that modifies N6-methyladenosine (m6A) on mRNA and regulates the translation of m6A-installed transcripts. m6A level on mRNA is increased in CLL cells with differential m6A highly enriched on splicing related transcripts. Moreover, high METTL3 expression in CLL is also associated with poor clinical outcomes. These results suggested that METTL3 translationally controls splicing factors through m6A and plays a role in CLL progression. Toward this end, we demonstrated that METTL3 is essential for CLL growth in both in vitro and in vivo studies. Knock out (KO) and pharmaceutical inhibition of METTL3 decreased cell growth and splicing factor expression. Overexpression of wildtype but not catalytic mutant METTL3 restored both defects in METTL3 KO cells, indicating that the regulation of splicing factor is m6A-dependent. To dissect the underlying mechanism, we performed an integrated Ribo-seq, RNA-seq, and MeRIP-seq on CLL cells with or without METTL3. KO of METTL3 decreased overall translation efficiency (TE) with RNA splicing as the most significantly affected pathway. Splicing factors with reduced TE displayed either hypo-m6A at stop codon region or hyper-m6A at CDS regions upon METTL3 KO, as direct or indirect targets of METTL3. Moreover, we found that m6A at stop codon and CDS regions regulates splicing factor translation via ribosome recycling and ribosome pausing, respectively. Taken together, our results uncovered a novel regulatory axis for METTL3 that controls splicing factor translation and contributes to CLL progression. Our study highlights a post-transcriptional layer of m6A modification as a major contributor to genetic lesion-independent splicing defects in CLL. Citation Format: Yiming Wu, Meiling Jin, Mike Fernandez, Kevyn Hart, Aijun Liao, Stacey M. Fernandes, Tinisha McDonald, Zhenhua Chen, Daniel Röth, Lucy Ghoda, Guido Marcucci, Markus Kalkum, Raju K. Pillai, Alexey V. Danilov, Jianjun Chen, Jennifer R. Brown, Steven T. Rosen, Tanya Siddiqi, Lili Wang. METTL3-mediated m6A modification controls splicing factor abundance and contributes to CLL progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3482.
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Exonic splicing enhancer
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RBM10, originally called S1‐1, is a nuclear RNA‐binding protein with domains characteristic of RNA processing proteins. It has been reported that RBM10 constitutes spliceosome complexes and that RBM5, a close homologue of RBM10, regulates alternative splicing of apoptosis‐related genes, Fas and cFLIP . In this study, we examined whether RBM10 has a regulatory function in splicing similar to RBM5, and determined that it indeed regulates alternative splicing of Fas and Bcl‐x genes. RBM10 promotes exon skipping of Fas pre‐mRNA as well as selection of an internal 5′‐splice site in Bcl‐x pre‐mRNA. We propose a consensus RBM10‐binding sequence at 5′‐splice sites of target exons and a mechanistic model of RBM10 action in the alternative splicing.
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Exonic splicing enhancer
Minigene
SR protein
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SUMMARY Human pre-mRNA splicing requires the removal of introns with highly variable lengths, from tens to over a million nucleotides. Therefore, mechanisms of intron recognition and splicing are likely not universal. Recently, we reported that splicing in a subset of human short introns with truncated polypyrimidine tracts depends on RBM17 (SPF45), instead of the canonical splicing factor U2AF heterodimer. Here, we demonstrate that SAP30BP, a factor previously implicated in transcriptional control, is an essential splicing cofactor for RBM17. In vitro binding and NMR analyses demonstrate that a U2AF-homology motif (UHM) in RBM17 binds directly to a newly identified UHM-ligand motif (ULM) in SAP30BP. We show that this RBM17–SAP30BP interaction is required to specifically recruit RBM17 to phosphorylated SF3B1 (SF3b155), a U2 snRNP component in active spliceosomes. We propose a unique mechanism for splicing in a subset of short introns, in which SAP30BP guides RBM17 in the assembly of active spliceosomes. Graphical abstract In brief Fukumura et al. discover a general splicing mechanism in a subset of human short introns with truncated polypyrimidine tracts. This splicing reaction is mediated by intermediary RBM17–SAP30BP complex, instead of the known U2AF heterodimer. SAP30BP binding to RBM17 may support RBM17 association with active phosphorylated SF3B1 in U2 snRNP. Highlights RBM17 (SPF45) is a splicing factor required for a subset of human short introns SAP30BP is an essential cofactor, which interacts with RBM17 via UHM–ULM binding RBM17 forms a weak complex with SAP30BP before its binding with SF3B1 in U2 snRNP RBM17–SAP30BP complex supports RBM17 to be recruited to active phosphorylated SF3B1
Spliceosome
snRNP
Splicing factor
Polypyrimidine tract
Exonic splicing enhancer
Minor spliceosome
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The spliceosome catalyzes pre-mRNA splicing in two steps. After catalytic step I, a major remodeling of the spliceosome occurs to establish the active site for step II. Here, we report the isolation of a cDNA encoding hSlu7, the human homolog of the yeast second step splicing factor Slu7. We show that hSlu7 associates with the spliceosome late in the splicing pathway, but at a stage prior to recognition of the 3' splice site for step II. In the absence of hSlu7, splicing is stalled between the catalytic steps in a novel complex, the CDeltahSlu7 complex. We provide evidence that this complex differs significantly in structure from the known spliceosomal complexes, yet is a functional intermediate between the catalytic steps of splicing. Together, our observations indicate that hSlu7 is required for a structural alteration of the spliceosome prior to the establishment of the catalytically active spliceosome for step II.
Spliceosome
Splicing factor
Polypyrimidine tract
Exonic splicing enhancer
Minor spliceosome
Prp24
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Pre-messenger RNA splicing involves multi-step assembly of the large spliceosome complexes that catalyse the two consecutive trans-esterification reactions, resulting in intron removal. There is evidence that proof-reading mechanisms monitor the fidelity of this complex process. Transcripts that fail these fidelity tests are thought to be directed to degradation pathways, permitting the splicing factors to be recycled. While studying the roles of splicing factors in vivo, in budding yeast, we performed targeted depletion of individual proteins, and analysed the effect on co-transcriptional spliceosome assembly and splicing efficiency. Unexpectedly, depleting factors such as Prp16 or Prp22, that are known to function at the second catalytic step or later in the splicing pathway, resulted in a defect in the first step of splicing, and accumulation of arrested spliceosomes. Through a kinetic analysis of newly synthesized RNA, we observed that a second step splicing defect (the primary defect) was rapidly followed by the first step of splicing defect. Our results show that knocking down a splicing factor can quickly lead to a recycling defect with splicing factors sequestered in stalled complexes, thereby limiting new rounds of splicing. We demonstrate that this 'feed-back' effect can be minimized by depleting the target protein more gradually or only partially, allowing a better separation between primary and secondary effects. Our findings indicate that splicing surveillance mechanisms may not always cope with spliceosome assembly defects, and suggest that work involving knock-down of splicing factors or components of other large complexes should be carefully monitored to avoid potentially misleading conclusions.
Spliceosome
Exonic splicing enhancer
Splicing factor
Protein splicing
Prp24
Polypyrimidine tract
Precursor mRNA
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Spliceosome
Splicing factor
Exonic splicing enhancer
Protein splicing
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Abstract Aberrant splicing is typically attributed to splice‐factor (SF) mutation and contributes to malignancies including acute myeloid leukemia (AML). Here, we discovered a mutation‐independent means to extensively reprogram alternative splicing (AS). We showed that the dysregulated expression of eukaryotic translation initiation factor eIF4E elevated selective splice‐factor production, thereby impacting multiple spliceosome complexes, including factors mutated in AML such as SF3B1 and U2AF1. These changes generated a splicing landscape that predominantly supported altered splice‐site selection for ~800 transcripts in cell lines and ~4,600 transcripts in specimens from high‐eIF4E AML patients otherwise harboring no known SF mutations. Nuclear RNA immunoprecipitations, export assays, polysome analyses, and mutational studies together revealed that eIF4E primarily increased SF production via its nuclear RNA export activity. By contrast, eIF4E dysregulation did not induce known SF mutations or alter spliceosome number. eIF4E interacted with the spliceosome and some pre‐mRNAs, suggesting its direct involvement in specific splicing events. eIF4E induced simultaneous effects on numerous SF proteins, resulting in a much larger range of splicing alterations than in the case of mutation or dysregulation of individual SFs and providing a novel paradigm for splicing control and dysregulation.
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EIF4E
Polypyrimidine tract
Eukaryotic translation
Initiation factor
Eukaryotic initiation factor
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Pre-mRNA splicing is performed by the spliceosome. SR proteins in this macromolecular complex are essential for both constitutive and alternative splicing. By using the SR-related protein ZNF265 as bait in a yeast two-hybrid screen, we pulled out the uncharacterized human protein XE7, which is encoded by a pseudoautosomal gene. XE7 had been identified in a large-scale proteomic analysis of the human spliceosome. It consists of two different isoforms produced by alternative splicing. The arginine/serine (RS)-rich region in the larger of these suggests a role in mRNA processing. Herein we show for the first time that XE7 is an alternative splicing regulator. XE7 interacts with ZNF265, as well as with the essential SR protein ASF/SF2. The RS-rich region of XE7 dictates both interactions. We show that XE7 localizes in the nucleus of human cells, where it colocalizes with both ZNF265 and ASF/SF2, as well as with other SR proteins, in speckles. We also demonstrate that XE7 influences alternative splice site selection of pre-mRNAs from CD44, Tra2-β1 and SRp20 minigenes. We have thus shown that the spliceosomal component XE7 resembles an SR-related splicing protein, and can influence alternative splicing.
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SR protein
Exonic splicing enhancer
Polypyrimidine tract
Protein splicing
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Different from canonical ubiquitin-like proteins, Hub1 does not form covalent conjugates with substrates but binds proteins non-covalently. In Saccharomyces cerevisiae, Hub1 associates with spliceosomes and mediates alternative splicing of SRC1, without affecting pre-mRNA splicing generally. Human Hub1 is highly similar to its yeast homolog, but its cellular function remains largely unexplored. Here, we show that human Hub1 binds to the spliceosomal protein Snu66 as in yeast; however, unlike its S. cerevisiae homolog, human Hub1 is essential for viability. Prolonged in vivo depletion of human Hub1 leads to various cellular defects, including splicing speckle abnormalities, partial nuclear retention of mRNAs, mitotic catastrophe, and consequently cell death by apoptosis. Early consequences of Hub1 depletion are severe splicing defects, however, only for specific splice sites leading to exon skipping and intron retention. Thus, the ubiquitin-like protein Hub1 is not a canonical spliceosomal factor needed generally for splicing, but rather a modulator of spliceosome performance and facilitator of alternative splicing.
Spliceosome
Exonic splicing enhancer
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Protein splicing
Polypyrimidine tract
Minor spliceosome
Exon skipping
Prp24
SR protein
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<p>Fig S1. SF3B1 mutant CLL samples had pervasive changes in 3’ splice site. Fig S2. Omics analyses identify widespread post-transcriptional upregulation of splicing factors in CLL. Fig S3. Abundance of spliceosome complexes and RNA-binding proteins are associated with clinical outcomes in CLL. Fig S4. METTL3 is consistently upregulated along with differential m6A modification on transcripts of RNA splicing process in CLL. Fig S5. KO or pharmacological inhibition of METTL3 impacts cell growth. Fig S6. KO or pharmacological inhibition of METTL3 impacts apoptosis, cell cycle, and splicing factor abundance. Fig S7. Splicing factors are either direct or indirect targets of METTL3. Fig S8. Validation of association between m6A and splicing factor abundance using dCasRx-METTL3.</p>
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