Role In Splicing Regulation Research Articles

Intronic small nucleolar RNAs regulate host gene splicing through base pairing with their adjacent intronic sequences

BackgroundSmall nucleolar RNAs (snoRNAs) are abundant noncoding RNAs best known for their involvement in ribosomal RNA maturation. In mammals, most expressed snoRNAs are embedded in introns of longer genes and produced through transcription and splicing of their host. Intronic snoRNAs were long viewed as inert passengers with little effect on host expression. However, a recent study reported a snoRNA influencing the splicing and ultimate output of its host gene. Overall, the general contribution of intronic snoRNAs to host expression remains unclear.ResultsComputational analysis of large-scale human RNA-RNA interaction datasets indicates that 30% of detected snoRNAs interact with their host transcripts. Many snoRNA-host duplexes are located near alternatively spliced exons and display high sequence conservation suggesting a possible role in splicing regulation. The study of the model SNORD2-EIF4A2 duplex indicates that the snoRNA interaction with the host intronic sequence conceals the branch point leading to decreased inclusion of the adjacent alternative exon. Extended SNORD2 sequence containing the interacting intronic region accumulates in sequencing datasets in a cell-type-specific manner. Antisense oligonucleotides and mutations that disrupt the formation of the snoRNA-intron structure promote the splicing of the alternative exon, shifting the EIF4A2 transcript ratio away from nonsense-mediated decay.ConclusionsMany snoRNAs form RNA duplexes near alternative exons of their host transcripts, placing them in optimal positions to control host output as shown for the SNORD2-EIF4A2 model system. Overall, our study supports a more widespread role for intronic snoRNAs in the regulation of their host transcript maturation.

FUS Alters circRNA Metabolism in Human Motor Neurons Carrying the ALS-Linked P525L Mutation.

Deregulation of RNA metabolism has emerged as one of the key events leading to the degeneration of motor neurons (MNs) in Amyotrophic Lateral Sclerosis (ALS) disease. Indeed, mutations on RNA-binding proteins (RBPs) or on proteins involved in aspects of RNA metabolism account for the majority of familiar forms of ALS. In particular, the impact of the ALS-linked mutations of the RBP FUS on many aspects of RNA-related processes has been vastly investigated. FUS plays a pivotal role in splicing regulation and its mutations severely alter the exon composition of transcripts coding for proteins involved in neurogenesis, axon guidance, and synaptic activity. In this study, by using in vitro-derived human MNs, we investigate the effect of the P525L FUS mutation on non-canonical splicing events that leads to the formation of circular RNAs (circRNAs). We observed altered levels of circRNAs in FUSP525L MNs and a preferential binding of the mutant protein to introns flanking downregulated circRNAs and containing inverted Alu repeats. For a subset of circRNAs, FUSP525L also impacts their nuclear/cytoplasmic partitioning, confirming its involvement in different processes of RNA metabolism. Finally, we assess the potential of cytoplasmic circRNAs to act as miRNA sponges, with possible implications in ALS pathogenesis.

Abstract 778: Dysregulation of alternative mRNA splicing by oncogenic KRAS in lung adenocarcinoma

Abstract Alternative mRNA splicing is dysregulated in many cancers including lung adenocarcinoma. These aberrant splicing events can sometimes be explained by mutations in splice sites or splicing factors. However, the majority of mis-splicing in cancers remains unexplained. We hypothesize that oncogenic signaling pathways play a role in regulating alternative splicing. In this study, we focus on mRNA splicing activity regulated by the Ras signaling cascade, which is frequently mutated in cancers. These pathways offer avenues for therapeutic modulation through small molecule inhibitors.We took a global proteomic and transcriptomic approach to study the effects of oncogenic Ras signaling on RNA splicing in vitro. Specifically, we performed LC-MS/MS and RNA-seq profiling on normal human lung airway epithelial (AALE) cells overexpressing wild-type or mutant KRAS or RIT1 (N = 3 replicates per allele). Phosphorylation of splicing factors was preferentially downregulated in KRASmut cells compared to KRASWT cells, suggesting that oncogenic KRAS signaling regulates splicing factor activity. Additionally, of the 2227 and 2452 skipped exon (SE) events in KRASG12V and KRASQ61H cells, respectively, compared to KRASWT overexpressing cells, 1013 events were shared between the two mutants. This statistically significant overlap (p < 0.001, hypergeometric test) indicates that KRAS has a role in splicing regulation that is disrupted when the protein is mutated. To determine how KRAS-regulated splicing activity compared to other signaling proteins and oncogenes, we also profiled the whole transcriptomes of isogenic A549 lung adenocarcinoma cell lines overexpressing 86 wild-type or variant alleles across 27 genes implicated in lung cancers (N = 4 to 8 replicates per allele). Of all pairs of mutant and wild-type alleles tested, KRASmut cells compared to KRASWT overexpressing cells exhibited the second highest levels of alternative splicing, behind only RNA binding protein RBM45 and its mutant allele RBM45M126I. In particular, ten SE events were observed to be differentially regulated in a KRAS dependent manner in both AALE and A549 cells. One of these 10 events is the differential splicing of the Myc Associated Zinc Finger (MAZ) protein to increase the expression of its splice variant that acts as a negative regulator of transcription. As MAZ regulates expression of KRAS, this splice variant may be a mechanism for the cell to modulate wild-type KRAS levels in the presence of KRAS mutants. Our proteomic and transcriptomic profiling in lung epithelial and lung adenocarcinoma cells uncovers splicing factor activity and mRNA splicing events regulated by oncogenic KRAS. With further studies of the alternative splicing events described, it will be possible to design or repurpose therapies in order to target aberrant splicing caused by disrupted Ras signaling and its downstream effectors. Citation Format: April Lo, Maria McSharry, Alice H. Berger. Dysregulation of alternative mRNA splicing by oncogenic KRAS in lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 778.

Single-molecule RNA remodelling by the DExH-Box 34 helicase

RNA helicases play essential roles in multiple cellular processes such as pre-mRNA splicing, mRNA translation and RNA quality-control. Amongst them, the DExH-Box proteins family comprises ATP-dependent RNA remodelers involved in several key aspects of these processes. In particular, the DExH-Box 34 (DHX34) RNA helicase is an essential protein involved in non-sense mediated decay (NMD) and has been suggested to have a role in splicing regulation. However, the precise molecular mechanism by which DHX34 functions remains largely unknown.

Wilms’ tumor 1-associating protein complex regulates alternative splicing and polyadenylation at potential G-quadruplex-forming splice site sequences

Wilms’ tumor 1-associating protein (WTAP) is a core component of the N6-methyladenosine (m6A)-methyltransferase complex, along with VIRMA, CBLL1, ZC3H13 (KIAA0853), RBM15/15B, and METTL3/14, which generate m6A, a key RNA modification that affects various processes of RNA metabolism. WTAP also interacts with splicing factors; however, despite strong evidence suggesting a role of Drosophila WTAP homolog fl(2)d in alternative splicing (AS), its role in splicing regulation in mammalian cells remains elusive. Here we demonstrate using RNAi coupled with RNA-seq that WTAP, VIRMA, CBLL1, and ZC3H13 modulate AS, promoting exon skipping and intron retention in AS events that involve short introns/exons with higher GC content and introns with weaker polypyrimidine-tract and branch points. Further analysis of GC-rich sequences involved in AS events regulated by WTAP, together with minigene assay analysis, revealed potential G-quadruplex formation at splice sites where WTAP has an inhibitory effect. We also found that several AS events occur in the last exon of one isoform of MSL1 and WTAP, leading to competition for polyadenylation. Proteomic analysis also suggested that WTAP/CBLL1 interaction promotes recruitment of the 3′-end processing complex. Taken together, our results indicate that the WTAP complex regulates AS and alternative polyadenylation via inhibitory mechanisms in GC-rich sequences.

CTCF as a regulator of alternative splicing: new tricks for an old player.

Three decades of research have established the CCCTC-binding factor (CTCF) as a ubiquitously expressed chromatin organizing factor and master regulator of gene expression. A new role for CTCF as a regulator of alternative splicing (AS) has now emerged. CTCF has been directly and indirectly linked to the modulation of AS at the individual transcript and at the transcriptome-wide level. The emerging role of CTCF-mediated regulation of AS involves diverse mechanisms; including transcriptional elongation, DNA methylation, chromatin architecture, histone modifications, and regulation of splicing factor expression and assembly. CTCF thereby appears to not only co-ordinate gene expression regulation but contributes to the modulation of transcriptomic complexity. In this review, we highlight previous discoveries regarding the role of CTCF in AS. In addition, we summarize detailed mechanisms by which CTCF mediates AS regulation. We propose opportunities for further research designed to examine the possible fate of CTCF-mediated alternatively spliced genes and associated biological consequences. CTCF has been widely acknowledged as the ‘master weaver of the genome’. Given its multiple connections, further characterization of CTCF’s emerging role in splicing regulation might extend its functional repertoire towards a ‘conductor of the splicing orchestra’.

Characterizing a cancer spliceosome

Cancer Genomics Cells undergo many genomic changes as they progress toward metastatic cancer. One aspect of this change is to RNA expression and splicing isoforms, but how these differences affect tumor progression is not well characterized. Fish et al. developed a computational framework called pyTEISER that identifies structural cis-regulatory elements that control diverse types of RNA regulation. Applying pyTEISER to models of breast cancer metastasis, they discovered an RNA short-stem-loop element that forms a “structural splicing enhancer” that acts in cis to regulate alternative splicing of RNA transcripts. One of these interactions encompasses the RNA-binding protein SNRPA1 and results in alternative exon inclusion that affects metastatic capacity in xenograft models. Thus, RNA element binding may play a role in splicing regulation and is potentially an important component of the cis-splicing code. Science , this issue p. [eabc7531][1] [1]: /lookup/doi/10.1126/science.abc7531

Germline PTEN mutations are associated with a skewed peripheral immune repertoire in humans and mice.

Individuals with germline mutations in the gene encoding phosphatase and tensin homolog on chromosome ten (PTEN) are diagnosed with PTEN hamartoma tumor syndrome (PHTS) and are at high risk for developing breast, thyroid and other cancers and/or autoimmunity or neurodevelopmental issues including autism spectrum disorders. Although well recognized as a tumor suppressor, involvement of PTEN mutations in mediating such a diverse range of phenotypes indicates a more central involvement for PTEN in immunity than previously recognized. To address this, sequencing of the T-cell receptor variable-region β-chain was performed on peripheral blood from PHTS patients. Based on patient findings, we performed mechanistic studies in two Pten knock-in murine models, distinct from each other in cell compartment-specific predominance of Pten. We found that PTEN mutations in humans and mice are associated with a skewed T- and B-cell gene repertoire, characterized by increased prevalence of high-frequency clones. Immunological characterization showed that Pten mutants have increased B-cell proliferation and a proclivity towards increased T-cell reactivity upon Toll-like-receptor stimulation. Furthermore, decreases in nuclear but not cytoplasmic Pten levels associated with a reduction in expression of the autoimmune regulator (Aire), a critical mediator of central immune tolerance. Mechanistically, we show that nuclear PTEN most likely regulates Aire expression via its emerging role in splicing regulation. We conclude that germline disruption of PTEN, both in human and mouse, results in compromised central immune tolerance processes that may significantly impact individual stress responses and therefore predisposition to autoimmunity and cancer.

Dengue virus targets RBM10 deregulating host cell splicing and innate immune response.

RNA-seq experiments previously performed by our laboratories showed enrichment in intronic sequences and alterations in alternative splicing in dengue-infected human cells. The transcript of the SAT1 gene, of well-known antiviral action, displayed higher inclusion of exon 4 in infected cells, leading to an mRNA isoform that is degraded by non-sense mediated decay. SAT1 is a spermidine/spermine acetyl-transferase enzyme that decreases the reservoir of cellular polyamines, limiting viral replication. Delving into the molecular mechanism underlying SAT1 pre-mRNA splicing changes upon viral infection, we observed lower protein levels of RBM10, a splicing factor responsible for SAT1 exon 4 skipping. We found that the dengue polymerase NS5 interacts with RBM10 and its sole expression triggers RBM10 proteasome-mediated degradation. RBM10 over-expression in infected cells prevents SAT1 splicing changes and limits viral replication, while its knock-down enhances the splicing switch and also benefits viral replication, revealing an anti-viral role for RBM10. Consistently, RBM10 depletion attenuates expression of interferon and pro-inflammatory cytokines. In particular, we found that RBM10 interacts with viral RNA and RIG-I, and even promotes the ubiquitination of the latter, a crucial step for its activation. We propose RBM10 fulfills diverse pro-inflammatory, anti-viral tasks, besides its well-documented role in splicing regulation of apoptotic genes.

Iterative random forests to discover predictive and stable high-order interactions

Genomics has revolutionized biology, enabling the interrogation of whole transcriptomes, genome-wide binding sites for proteins, and many other molecular processes. However, individual genomic assays measure elements that interact in vivo as components of larger molecular machines. Understanding how these high-order interactions drive gene expression presents a substantial statistical challenge. Building on random forests (RFs) and random intersection trees (RITs) and through extensive, biologically inspired simulations, we developed the iterative random forest algorithm (iRF). iRF trains a feature-weighted ensemble of decision trees to detect stable, high-order interactions with the same order of computational cost as the RF. We demonstrate the utility of iRF for high-order interaction discovery in two prediction problems: enhancer activity in the early Drosophila embryo and alternative splicing of primary transcripts in human-derived cell lines. In Drosophila, among the 20 pairwise transcription factor interactions iRF identifies as stable (returned in more than half of bootstrap replicates), 80% have been previously reported as physical interactions. Moreover, third-order interactions, e.g., between Zelda (Zld), Giant (Gt), and Twist (Twi), suggest high-order relationships that are candidates for follow-up experiments. In human-derived cells, iRF rediscovered a central role of H3K36me3 in chromatin-mediated splicing regulation and identified interesting fifth- and sixth-order interactions, indicative of multivalent nucleosomes with specific roles in splicing regulation. By decoupling the order of interactions from the computational cost of identification, iRF opens additional avenues of inquiry into the molecular mechanisms underlying genome biology.

Molecular Mechanisms for CFIm-Mediated Regulation of mRNA Alternative Polyadenylation

Alternative mRNA processing is a critical mechanism for proteome expansion and gene regulation in higher eukaryotes. The SR family proteins play important roles in splicing regulation. Intriguingly, mammalian genomes encode many poorly characterized SR-like proteins, including subunits of the mRNA 3'-processing factor CFIm, CFIm68 and CFIm59. Here we demonstrate that CFIm functions as an enhancer-dependent activator of mRNA 3' processing. CFIm regulates global alternative polyadenylation (APA) by specifically binding and activating enhancer-containing poly(A) sites (PASs). Importantly, the CFIm activator functions are mediated by the arginine-serine repeat (RS) domains of CFIm68/59, which bind specifically to an RS-like region in the CPSF subunit Fip1, and this interaction is inhibited by CFIm68/59 hyper-phosphorylation. The remarkable functional similarities between CFIm and SR proteins suggest that interactions between RS-like domains in regulatory and core factors may provide a common activation mechanism for mRNA 3' processing, splicing, and potentially other steps in RNA metabolism.

Human METTL16 is a N6-methyladenosine (m6A) methyltransferase that targets pre-mRNAs and various non-coding RNAs.

N6-methyladenosine (m6A) is a highly dynamic RNA modification that has recently emerged as a key regulator of gene expression. While many m6A modifications are installed by the METTL3-METTL14 complex, others appear to be introduced independently, implying that additional human m6A methyltransferases remain to be identified. Using crosslinking and analysis of cDNA (CRAC), we reveal that the putative human m6A "writer" protein METTL16 binds to the U6 snRNA and other ncRNAs as well as numerous lncRNAs and pre-mRNAs. We demonstrate that METTL16 is responsible for N6-methylation of A43 of the U6 snRNA and identify the early U6 biogenesis factors La, LARP7 and the methylphosphate capping enzyme MEPCE as METTL16 interaction partners. Interestingly, A43 lies within an essential ACAGAGA box of U6 that base pairs with 5' splice sites of pre-mRNAs during splicing, suggesting that METTL16-mediated modification of this site plays an important role in splicing regulation. The identification of METTL16 as an active m6A methyltransferase in human cells expands our understanding of the mechanisms by which the m6A landscape is installed on cellular RNAs.

A compensatory mutagenesis study of a conserved hairpin in the M gene segment of influenza A virus shows its role in virus replication

ABSTRACTRNA structures are increasingly recognized to be of importance during influenza A virus replication. Here, we investigated a predicted conserved hairpin in the M gene segment (nt 967-994) within the region of the vRNA 5′ packaging signal. The existence of this RNA structure and its possible role in virus replication was investigated using a compensatory mutagenesis approach. Mutations were introduced in the hairpin stem, based on natural variation. Virus replication properties were studied for the mutant viruses with disrupted and restored RNA structures. Viruses with structure-disrupting mutations had lower virus titers and a significantly reduced median plaque size when compared with the wild-type (WT) virus, while viruses with structure restoring-mutations replicated comparable to WT. Moreover, virus replication was also reduced when mutations were introduced in the hairpin loop, suggesting its involvement in RNA interactions. Northern blot and FACS experiments were performed to study differences in RNA levels as well as production of M1 and M2 proteins, expressed via alternative splicing. Stem-disruptive mutants caused lower vRNA and M2 mRNA levels and reduced M2 protein production at early time-points. When the RNA structure was restored, vRNA, M2 mRNA and M2 protein levels were increased, demonstrating a compensatory effect. Thus, this study provides evidence for functional importance of the predicted M RNA structure and suggests its role in splicing regulation.

Biochemical Investigation into Novel Activities of Yeast SPOUT‐Superfamily RNA Methyltransferases

The SPOUT superfamily of S‐adenosyl‐L‐methionine (SAM) dependent methyltransferases are involved in post‐transcriptional RNA modifications. Nep1, a SPOUT superfamily member, methylates rRNA at the N1 position of a post‐transcriptionally incorporated pseudouridine in the 18S rRNA of Saccharomyces cerevisiae (yeast) and in humans is implicated in cases of Diamond‐Blackfan anemia. We found that Nep1 can bind and methylate a specific pseudouridine found in the U2 snRNA in vitro, suggesting that Nep1 may also play a role in splicing regulation. Given this finding, other SPOUT proteins may also be able to modify snRNAs and be involved in splicing regulation. YGR283C and YMR310C are two other yeast SPOUT methyl transferases whose specific biological functions are not known but are known to interact with the U2 snRNP yeast protein, Lea1, and the yeast homolog of dyskerin, a protein involved in pseudouridylation of the U2 snRNA. We have expressed yeast YGR283C in E. coli and purified the protein for biochemical and structural characterization. Fluorescence polarization assays and electrophoretic mobility shift assays will be conducted to quantify YGR283C binding affinities for snRNAs. Filter binding assays will be used to quantify [3H]‐methyl incorporation from SAM into snRNA substrates. Together these studies will elucidate the function of these previously uncharacterized SPOUT superfamily members.

Abstract B58: Single-nucleotide polymorphisms of race-related alternatively spliced genes associate with prostate cancer risk, aggressiveness and/or survival

Abstract Background: African American (AA) men exhibit nearly 2-fold higher incidence and 3-fold higher mortality rates from prostate cancer (PC) compared with white men. This disparity likely results from a complex interplay between behavioral, social, neighborhood and biological factors, which all work collectively to generate increased tumor aggressiveness in AAs. Recent data from our laboratory, evaluating human PC biopsy tissue led to the identification of alternative splicing events between AA and white PC that track with increased growth and more aggressive invasion characteristics of PC in AA men. In the present study, we explored associations between genetic variants of 30 such alternatively spliced genes and PC risk, aggressiveness and survival in white and AA groups by analyzing published genome-wide association studies (GWAS) of PC. Methods: We used GWAS datasets from the Multiethnic Cohort Study of Diet and Cancer (MEC), including AA PC cases and controls, and the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO), including white PC cases and controls, to evaluate associations of 11,073 and 10,385 single-nucleotide polymorphisms (SNPs), respectively, in 30 genes identified to be alternatively spliced between white and AA PC with PC risk, aggressiveness and survival. For risk, we evaluated 1150 cases and 1101 controls in PLCO and 670 cases and 658 controls in MEC, for aggressiveness, we evaluated 237 aggressive and 843 non-aggressive in PLCO and 234 aggressive and 436 non-aggressive in MEC, and for survival, we evaluated 1150 overall, 237 aggressive and 843 non-aggressive in PLCO. We then performed in silico bioinformatics to investigate potential functions of the SNPs. Results: Significant associations between SNPs in FN1, COL6A3 and ACACA and SNPs in SEMA3C and FASN and PC risk in white and AA populations, respectively, were identified. In addition, SNPs in ACACA and SNPs in SEMA3C, RELN, MYBPC1, NCOR2 and WDR4 were found to be significantly associated with PC aggressiveness in white and AA populations, respectively. Furthermore, significant associations between SNPs in RHOU, FN1, COL6A3, SEMA3C, RELN, CD44, LMO7 and WDR4 and PC survival in a white population were identified. All of the aforementioned SNPs were predicted to play a role in splicing regulation. Conclusions: SNPs of race-related alternatively spliced genes that are predicted to play a role in splicing regulation are significantly associated with PC risk, aggressiveness and/or survival in white and/or AA populations. Such variants have the potential to serve as novel molecular targets for development of biomarkers of increased risk of aggressive PC or therapeutics against aggressive PC. Ultimately, such biomarkers and therapeutic agents could serve as novel precision medicine interventions, reducing the mortality burden from PC among AA men. Citation Format: Jennifer Freedman, Yanru Wang, Hongliang Liu, Patricia Moorman, Terry Hyslop, Daniel George, Norman Lee, Qingyi Wei, Steven Patierno. Single-nucleotide polymorphisms of race-related alternatively spliced genes associate with prostate cancer risk, aggressiveness and/or survival. [abstract]. In: Proceedings of the Ninth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2016 Sep 25-28; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2017;26(2 Suppl):Abstract nr B58.

RBM4 Regulates Neuronal Differentiation of Mesenchymal Stem Cells by Modulating Alternative Splicing of Pyruvate Kinase M.

RBM4 promotes differentiation of neuronal progenitor cells and neurite outgrowth of cultured neurons via its role in splicing regulation. In this study, we further explored the role of RBM4 in neuronal differentiation. During neuronal differentiation, energy production shifts from glycolysis to oxidative phosphorylation. We found that the splice isoform change of the metabolic enzyme pyruvate kinase M (PKM) from PKM2 to PKM1 occurs during brain development and is impaired in RBM4-deficient brains. The PKM isoform change could be recapitulated in human mesenchymal stem cells (MSCs) during neuronal induction. Using a PKM minigene, we demonstrated that RBM4 plays a direct role in regulating alternative splicing of PKM. Moreover, RBM4 antagonized the function of the splicing factor PTB and induced the expression of a PTB isoform with attenuated splicing activity in MSCs. Overexpression of RBM4 or PKM1 induced the expression of neuronal genes, increased the mitochondrial respiration capacity in MSCs, and, accordingly, promoted neuronal differentiation. Finally, we demonstrated that RBM4 is induced and is involved in the PKM splicing switch and neuronal gene expression during hypoxia-induced neuronal differentiation. Hence, RBM4 plays an important role in the PKM isoform switch and the change in mitochondrial energy production during neuronal differentiation.

The RNA-binding profile of Acinus, a peripheral component of the exon junction complex, reveals its role in splicing regulation.

Acinus (apoptotic chromatin condensation inducer in the nucleus) is an RNA-binding protein (RBP) originally identified for its role in apoptosis. It was later found to be an auxiliary component of the exon junction complex (EJC), which is deposited at exon junctions as a consequence of pre-mRNA splicing. To uncover the cellular functions of Acinus and investigate its role in splicing, we mapped its endogenous RNA targets using the cross-linking immunoprecipitation protocol (iCLIP). We observed that Acinus binds to pre-mRNAs, associating specifically to a subset of suboptimal introns, but also to spliced mRNAs. We also confirmed the presence of Acinus as a peripheral factor of the EJC. RNA-seq was used to investigate changes in gene expression and alternative splicing following siRNA-mediated depletion of Acinus in HeLa cells. This analysis revealed that Acinus is preferentially required for the inclusion of specific alternative cassette exons and also controls the faithful splicing of a subset of introns. Moreover, a large number of splicing changes can be related to Acinus binding, suggesting a direct role of Acinus in exon and intron definition. In particular, Acinus regulates the splicing of DFFA/ICAD transcript, a major regulator of DNA fragmentation. Globally, the genome-wide identification of RNA targets of Acinus revealed its role in splicing regulation as well as its involvement in other cellular pathways, including cell cycle progression. Altogether, this study uncovers new cellular functions of an RBP transiently associated with the EJC.

High-throughput analyses of hnRNP H1 dissects its multi-functional aspect

ABSTRACThnRNPs are polyvalent RNA binding proteins that have been implicated in a range of regulatory roles including splicing, mRNA decay, translation, and miRNA metabolism. A variety of genome wide studies have taken advantage of methods like CLIP and RIP to identify the targets and binding sites of RNA binding proteins. However, due to the complex nature of RNA-binding proteins, these studies are incomplete without assays that characterize the impact of RBP binding on mRNA target expression. Here we used a suite of high-throughput approaches (RIP-Seq, iCLIP, RNA-Seq and shotgun proteomics) to provide a comprehensive view of hnRNP H1s ensemble of targets and its role in splicing, mRNA decay, and translation. The combination of RIP-Seq and iCLIP allowed us to identify a set of 1,086 high confidence target transcripts. Binding site motif analysis of these targets suggests the TGGG tetramer as a prevalent component of hnRNP H1 binding motif, with particular enrichment around intronic hnRNP H1 sites. Our analysis of the target transcripts and binding sites indicates that hnRNP H1s involvement in splicing is 2-fold: it directly affects a substantial number of splicing events, but also regulates the expression of major components of the splicing machinery and other RBPs with known roles in splicing regulation. The identified mRNA targets displayed function enrichment in MAPK signaling and ubiquitin mediated proteolysis, which might be main routes by which hnRNP H1 promotes tumorigenesis.

Global regulation of alternative splicing by adenosine deaminase acting on RNA (ADAR)

Alternative mRNA splicing is a major mechanism for gene regulation and transcriptome diversity. Despite the extent of the phenomenon, the regulation and specificity of the splicing machinery are only partially understood. Adenosine-to-inosine (A-to-I) RNA editing of pre-mRNA by ADAR enzymes has been linked to splicing regulation in several cases. Here we used bioinformatics approaches, RNA-seq and exon-specific microarray of ADAR knockdown cells to globally examine how ADAR and its A-to-I RNA editing activity influence alternative mRNA splicing. Although A-to-I RNA editing only rarely targets canonical splicing acceptor, donor, and branch sites, it was found to affect splicing regulatory elements (SREs) within exons. Cassette exons were found to be significantly enriched with A-to-I RNA editing sites compared with constitutive exons. RNA-seq and exon-specific microarray revealed that ADAR knockdown in hepatocarcinoma and myelogenous leukemia cell lines leads to global changes in gene expression, with hundreds of genes changing their splicing patterns in both cell lines. This global change in splicing pattern cannot be explained by putative editing sites alone. Genes showing significant changes in their splicing pattern are frequently involved in RNA processing and splicing activity. Analysis of recently published RNA-seq data from glioblastoma cell lines showed similar results. Our global analysis reveals that ADAR plays a major role in splicing regulation. Although direct editing of the splicing motifs does occur, we suggest it is not likely to be the primary mechanism for ADAR-mediated regulation of alternative splicing. Rather, this regulation is achieved by modulating trans-acting factors involved in the splicing machinery.

Regulating the regulators: Serine/arginine‐rich proteins under scrutiny

Serine/arginine-rich (SR) proteins are among the most studied splicing regulators. They constitute a family of evolutionarily conserved proteins that, apart from their initially identified and deeply studied role in splicing regulation, have been implicated in genome stability, chromatin binding, transcription elongation, mRNA stability, mRNA export and mRNA translation. Remarkably, this list of SR protein activities seems far from complete, as unexpected functions keep being unraveled. An intriguing aspect that awaits further investigation is how the multiple tasks of SR proteins are concertedly regulated within mammalian cells. In this article, we first discuss recent findings regarding the regulation of SR protein expression, activity and accessibility. We dive into recent studies describing SR protein auto-regulatory feedback loops involving different molecular mechanisms such asunproductive splicing, microRNA-mediated regulation and translational repression. In addition, we take into account another step of regulation of SR proteins, presenting new findings about a variety of post-translational modifications by proteomics approaches and how some of these modifications can regulate SR protein sub-cellular localization or stability. Towards the end, we focus in two recently revealed functions of SR proteins beyond mRNA biogenesis and metabolism, the regulation of micro-RNA processing and the regulation of small ubiquitin-like modifier (SUMO) conjugation.