Modelling splicing

L’Splicing de les molècules d’ARN és el procés pel qual les seqüències interposades (“introns”) s’eliminen, i les seqüències restants es concatenen per a formar l’ARN madur. La investigació recent mostra que gairebé tots els gens amb splicing es veuen afectats per splicing alternatiu. Aquí, en primer lloc definim la longitud mínima d’un oligomer d’ARN per a funcionar com a lloc d’unió d’un factor d’splicing. A continuació, explorem la capacitat d’aquests oligomers per a predir estructures completes exó-intró. Destaquem els oligomers que són més informatius per a això, i demostrem que la mateixa precisió com en enfocaments anteriors es pot aconseguir amb menys oligomers. L’observació de que aquest enfocament és lluny de predir amb exactitud tota l’estructura exó-intró ens va portar a investigar els factors que juguen un paper en l’splicing co-transcripcional. Demostrem que els nucleosomes es col.loquen preferentment en els exons i plantegem la hipòtesi que juguen un paper en les decisions de l’splicing. A continuació, introduïm el “completed splicing index” i concluem que l’splicing co-transcripcional és molt generalitzat. A més, l’splicing co-transcripcional mostra vincles amb l’organització de la cromatina. A la llum d’aquests resultats, es van supervisar els canvis de la cromatina en exons diferencialment inclosos en dos teixits. Hem descobert una varietat de marques de les histones, però no totes, mostrant un comportament significativament diferent en els exons més inclosos i més exclosos. Las marques més destacades que apareixen són H3K9ac i dos estats de metilació de lisina 4.Splicing of RNA molecules is the process, by which intervening sequences (“introns”) in the primary transcript are excised, and the remaining sequences (“exons”) are concatenated to form the mature RNA. Recent evidence shows that almost all spliced genes are affected by alternative splicing. Here, we define the minimal length of RNA oligomers that can sensibly be called splicing factor binding sites. Then, we explore the capacity of these oligomers to predict complete exon-intron structures. We highlight those oligomers that are most informative for this and show, that equal accuracy as in previous approaches can be achieved with less RNA oligomers. The observation, that this approach falls short of accurately predicting the entire exon-intron structure, led us to investigate determinants linked to co-transcriptional splicing. We show that nucleosomes are preferentially positioned on exons and hypothesize that they play a role in splicing decisions. We then introduce the “completed splicing index” and conclude that co-transcriptional splicing is very wide-spread in humans. Furthermore co-transcriptional splicing exhibits links to chromatin organization. In the light of these results, we go on to monitor chromatin changes on differentially included exons in pair-wise tissue comparisons. We find a variety of histone marks, but not all, showing significantly different behavior on up- and downregulated exons. The most prominently appearing marks are H3K9ac and two lysine 4 methylation states

From chromatin to splicing: RNA-processing as a total artwork

RNA plays a central role in the determination of the phenotype of the cell. The molecular mechanisms involved in primary RNA synthesis and subsequent post-processing are not completely understood, but there is increasing evidence that they are more tightly coupled than previously expected. The analyses by a number of groups of recently published genome wide maps of chromatin structure have further uncovered a role for primary chromatin structure in RNA processing. Indeed, these analyses have revealed that nucleosomes show a characteristic occupancy pattern in exonic regions of metazoan genomes. The pattern is strongly indicative of an implication of nucleosome positioning in exon recognition during pre-mRNA splicing. Characteristic exonic patterns have also been observed for a number of histone modifications, suggesting the possibility that chromatin state plays a direct role in the regulation of splicin

Erratum to: Promoter-like epigenetic signatures in exons displaying cell type-specific splicing

After the publication of this work [1] an error was noticed in Fig. 7. The panel‘h’ is missing from Fig. 7. Please see the corrected figure below. The publisher apologises for this error

Promoter-like epigenetic signatures in exons displaying cell type-specific splicing

Background. Pre-mRNA splicing occurs mainly co-transcriptionally, and both nucleosome density and histone modifications have been proposed to play a role in splice site recognition and regulation. However, the extent and mechanisms behind this interplay remain poorly understood./nResults. We use transcriptomic and epigenomic data generated by the ENCODE project to investigate the association between chromatin structure and alternative splicing. We find a strong and significant positive association between H3K9ac, H3K27ac, H3K4me3, epigenetic marks characteristic of active promoters, and exon inclusion in a small but well-defined class of exons, representing approximately 4 % of all regulated exons. These exons are systematically maintained at comparatively low levels of inclusion across cell types, but their inclusion is significantly enhanced in particular cell types when in physical proximity to active promoters./nConclusion. Histone modifications and other chromatin features that activate transcription can be co-opted to participate in the regulation of the splicing of exons that are in physical proximity to promoter regions.We acknowledge support of the Spanish Ministry of Economy and Competitiveness, ‘Centro de Excelencia Severo Ochoa 2013-2017’, SEV-2012-0208. JC was supported by a SFRH/BD/33535/2008 from the Portuguese Foundation to Science and Technology. CI was supported by a La Caixa predoctoral fellowship. Work in JV’s lab was supported by Fundación Botín, by Banco de Santander through its Santander Universities Global Division and by Consolider RNAREG, MINECO, and AGAUR. We thank Anshul Kundaje, Ben Brown, Michael Snyder, Thomas Gingeras, and Alberto Kornblihtt for useful discussions and access to data, and Romina Garrido for administrative assistance

Role of six single nucleotide polymorphisms, risk factors in coronary disease, in OLR1 alternative splicing

The OLR1 gene encodes the oxidized low-density lipoprotein receptor (LOX-1), which is responsible for the cellular uptake of oxidized LDL (Ox-LDL), foam cell formation in atheroma plaques and atherosclerotic plaque rupture. Alternative splicing (AS) of OLR1 exon 5 generates two protein isoforms with antagonistic functions in Ox-LDL uptake. Previous work identified six single nucleotide polymorphisms (SNPs) in linkage disequilibrium that influence the inclusion levels of OLR1 exon 5 and correlate with the risk of cardiovascular disease. Here we use minigenes to recapitulate the effects of two allelic series (Low- and High-Risk) on OLR1 AS and identify one SNP in intron 4 (rs3736234) as the main contributor to the differences in exon 5 inclusion, while the other SNPs in the allelic series attenuate the drastic effects of this key SNP. Bioinformatic, proteomic, mutational and functional high-throughput analyses allowed us to define regulatory sequence motifs and identify SR protein family members (SRSF1, SRSF2) and HMGA1 as factors involved in the regulation of OLR1 AS. Our results suggest that antagonism between SRSF1 and SRSF2/HMGA1, and differential recognition of their regulatory motifs depending on the identity of the rs3736234 polymorphism, influence OLR1 exon 5 inclusion and the efficiency of Ox-LDL uptake, with potential implications for atherosclerosis and coronary disease

Role of six single nucleotide polymorphisms, risk factors in coronary disease, in OLR1 alternative splicing

The OLR1 gene encodes the oxidized low-density lipoprotein receptor (LOX-1), which is responsible for the cellular uptake of oxidized LDL (Ox-LDL), foam cell formation in atheroma plaques and atherosclerotic plaque rupture. Alternative splicing (AS) of OLR1 exon 5 generates two protein isoforms with antagonistic functions in Ox-LDL uptake. Previous work identified six single nucleotide polymorphisms (SNPs) in linkage disequilibrium that influence the inclusion levels of OLR1 exon 5 and correlate with the risk of cardiovascular disease. Here we use minigenes to recapitulate the effects of two allelic series (Low- and High-Risk) on OLR1 AS and identify one SNP in intron 4 (rs3736234) as the main contributor to the differences in exon 5 inclusion, while the other SNPs in the allelic series attenuate the drastic effects of this key SNP. Bioinformatic, proteomic, mutational and functional high-throughput analyses allowed us to define regulatory sequence motifs and identify SR protein family members (SRSF1, SRSF2) and HMGA1 as factors involved in the regulation of OLR1 AS. Our results suggest that antagonism between SRSF1 and SRSF2/HMGA1, and differential recognition of their regulatory motifs depending on the identity of the rs3736234 polymorphism, influence OLR1 exon 5 inclusion and the efficiency of Ox-LDL uptake, with potential implications for atherosclerosis and coronary disease

Absence of canonical marks of active chromatin in developmentally regulated genes

The interplay of active and repressive histone modifications is assumed to have a key role in the regulation of gene expression. In contrast to this generally accepted view, we show that the transcription of genes temporally regulated during fly and worm development occurs in the absence of canonically active histone modifications. Conversely, strong chromatin marking is related to transcriptional and post-transcriptional stability, an association that we also observe in mammals. Our results support a model in which chromatin marking is associated with the stable production of RNA, whereas unmarked chromatin would permit rapid gene activation and deactivation during development. In the latter case, regulation by transcription factors would have a comparatively more important regulatory role than chromatin marks.We thank the modENCODE project, the ENCODE Project (human and mouse data) and the Roadmap Epigenomics Mapping Consortium for granting open access of these resources to the scientific community. We also thank the Ultrasequencing Unit of the Centre for Genomic Regulation (CRG, Barcelona, Spain) for sample processing and the Confocal Unit of CCiTUB (Centres Científics i Tecnològics de la Universitat de Barcelona) (Universitat de Barcelona, Barcelona, Spain). This work was performed under the financial support of the Spanish Ministry of Economy and Competitiveness with grants BIO2011-26205 to R.G., CSD2007-00008 and BFU2012-36888 to M.C., and 'Centro de Excelencia Severo Ochoa 2013–2017', SEV-2012-0208 and the European Research Council/European Community's Seventh Framework Programme with grant 294653 RNA-MAPS to R.G. E.B. is supported by the European Commission's Seventh Framework Programme 4DCellFate grant 277899. J.C. is supported by grant SFRH/BD/33535/2008 from the Portuguese Foundation of Science and Technolog

Absence of canonical marks of active chromatin in developmentally regulated genes

The interplay of active and repressive histone modifications is assumed to have a key role in the regulation of gene expression. In contrast to this generally accepted view, we show that the transcription of genes temporally regulated during fly and worm development occurs in the absence of canonically active histone modifications. Conversely, strong chromatin marking is related to transcriptional and post-transcriptional stability, an association that we also observe in mammals. Our results support a model in which chromatin marking is associated with the stable production of RNA, whereas unmarked chromatin would permit rapid gene activation and deactivation during development. In the latter case, regulation by transcription factors would have a comparatively more important regulatory role than chromatin marks.We thank the modENCODE project, the ENCODE Project (human and mouse data) and the Roadmap Epigenomics Mapping Consortium for granting open access of these resources to the scientific community. We also thank the Ultrasequencing Unit of the Centre for Genomic Regulation (CRG, Barcelona, Spain) for sample processing and the Confocal Unit of CCiTUB (Centres Científics i Tecnològics de la Universitat de Barcelona) (Universitat de Barcelona, Barcelona, Spain). This work was performed under the financial support of the Spanish Ministry of Economy and Competitiveness with grants BIO2011-26205 to R.G., CSD2007-00008 and BFU2012-36888 to M.C., and 'Centro de Excelencia Severo Ochoa 2013–2017', SEV-2012-0208 and the European Research Council/European Community's Seventh Framework Programme with grant 294653 RNA-MAPS to R.G. E.B. is supported by the European Commission's Seventh Framework Programme 4DCellFate grant 277899. J.C. is supported by grant SFRH/BD/33535/2008 from the Portuguese Foundation of Science and Technolog

Deep sequencing of subcellular RNA fractions shows splicing to be predominantly co-transcriptional in the human genome but inefficient for lncRNAs

Splicing remains an incompletely understood process. Recent findings suggest that chromatin structure participates in its regulation. Here, we analyze the RNA from subcellular fractions obtained through RNA-seq in the cell line K562. We show that in the human genome, splicing occurs predominantly during transcription. We introduce the coSI measure, based on RNA-seq reads mapping to exon junctions and borders, to assess the degree of splicing completion around internal exons. We show that, as expected, splicing is almost fully completed in cytosolic polyA+ RNA. In chromatin-associated RNA (which includes the RNA that is being transcribed), for 5.6% of exons, the removal of the surrounding introns is fully completed, compared with 0.3% of exons for which no intron-removal has occurred. The remaining exons exist as a mixture of spliced and fewer unspliced molecules, with a median coSI of 0.75. Thus, most RNAs undergo splicing while being transcribed: "co-transcriptional splicing." Consistent with co-transcriptional spliceosome assembly and splicing, we have found significant enrichment of spliceosomal snRNAs in chromatin-associated RNA compared with other cellular RNA fractions and other nonspliceosomal snRNAs. CoSI scores decrease along the gene, pointing to a "first transcribed, first spliced" rule, yet more downstream exons carry other characteristics, favoring rapid, co-transcriptional intron removal. Exons with low coSI values, that is, in the process of being spliced, are enriched with chromatin marks, consistent with a role for chromatin in splicing during transcription. For alternative exons and long noncoding RNAs, splicing tends to occur later, and the latter might remain unspliced in some cases.This work has been carried out under grants RD07/0067/0012, BIO2006-03380, and CSD2007-00050 from the Spanish Ministry of Science, and grants 1U54HG004557-01 and 1U54HG004555-01 from the National Institutes of Healt

Deep sequencing of subcellular RNA fractions shows splicing to be predominantly co-transcriptional in the human genome but inefficient for lncRNAs

Splicing remains an incompletely understood process. Recent findings suggest that chromatin structure participates in its regulation. Here, we analyze the RNA from subcellular fractions obtained through RNA-seq in the cell line K562. We show that in the human genome, splicing occurs predominantly during transcription. We introduce the coSI measure, based on RNA-seq reads mapping to exon junctions and borders, to assess the degree of splicing completion around internal exons. We show that, as expected, splicing is almost fully completed in cytosolic polyA+ RNA. In chromatin-associated RNA (which includes the RNA that is being transcribed), for 5.6% of exons, the removal of the surrounding introns is fully completed, compared with 0.3% of exons for which no intron-removal has occurred. The remaining exons exist as a mixture of spliced and fewer unspliced molecules, with a median coSI of 0.75. Thus, most RNAs undergo splicing while being transcribed: "co-transcriptional splicing." Consistent with co-transcriptional spliceosome assembly and splicing, we have found significant enrichment of spliceosomal snRNAs in chromatin-associated RNA compared with other cellular RNA fractions and other nonspliceosomal snRNAs. CoSI scores decrease along the gene, pointing to a "first transcribed, first spliced" rule, yet more downstream exons carry other characteristics, favoring rapid, co-transcriptional intron removal. Exons with low coSI values, that is, in the process of being spliced, are enriched with chromatin marks, consistent with a role for chromatin in splicing during transcription. For alternative exons and long noncoding RNAs, splicing tends to occur later, and the latter might remain unspliced in some cases.This work has been carried out under grants RD07/0067/0012, BIO2006-03380, and CSD2007-00050 from the Spanish Ministry of Science, and grants 1U54HG004557-01 and 1U54HG004555-01 from the National Institutes of Healt