Alternative splicing was discovered simultaneously with splicing over three decades ago. Since then, an enormous body of evidence has demonstrated the prevalence of alternative splicing in multicellular eukaryotes, its key roles in determining tissue- and species-specific differentiation patterns, the multiple post- and co-transcriptional regulatory mechanisms that control it, and its causal role in hereditary disease and cancer. The emerging evidence places alternative splicing in a central position in the flow of eukaryotic genetic information, between transcription and translation, in that it can respond not only to various signalling pathways that target the splicing machinery but also to transcription factors and chromatin structure.

PRMT5Premature Stop Codon (prmt5-5) PRMT5 PRMT5Premature

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.

Light is a source of energy and also a regulator of plant physiological adaptations. We show here that light/dark conditions affect alternative splicing of a subset of Arabidopsis genes preferentially encoding proteins involved in RNA processing. The effect requires functional chloroplasts and is also observed in roots when the communication with the photosynthetic tissues is not interrupted, suggesting that a signaling molecule travels through the plant. Using photosynthetic electron transfer inhibitors with different mechanisms of action we deduce that the reduced pool of plastoquinones initiates a chloroplast retrograde signaling that regulates nuclear alternative splicing and is necessary for proper plant responses to varying light conditions.Light regulates approximately 20% of the transcriptome in Arabidopsis thaliana and rice (1, 2). Alternative splicing has been shown to modulate gene expression during plant development and in response to environmental cues (3). We observed that the alternative splicing of At-RS31 ( Figure 1A), encoding a Ser-Arg-rich splicing factor (4), changed in different light regimes, which led us to investigate how light regulates alternative splicing in plants. Figure 1B). This effect was rapidly reversed when seedlings were placed back in light, with total recovery of the original SI in about 3 hr ( Figure 1C), indicating that the kinetics of the splicing response is slower from light to dark than from dark to light.The light effect is gene-specific ( Figure S1) and is also observed in diurnal cycles under short day conditions ( Figures 1D and S2). Furthermore, three circadian clock mutants behaved like the wild type (WT) in the response of At-RS31 alternative splicing to light/dark ( Figure S3). Changes in At-RS31 splicing are proportional to light intensity both under constant light or in short day grown seedlings ( Figure S4).Both red (660 nm) and blue (470 nm) lights produced similar results as white light ( Figure 1E). Moreover, At-RS31 splicing is not affected in phytochrome and cryptochrome signaling mutants (5, 6) behave as WT seedlings, ruling out photosensory pathways in this light regulation (Figures 1F, S5 and S6).Light-triggered changes in At-RS31 mRNA patterns are not due to differential mRNA degradation. First, the light effect is not observed in the presence of the transcription inhibitor actinomycin D ( Figure 1G). Second, the effects are still observed in upf mutants, defective in the nonsense-mediated mRNA decay (NMD) pathway (7) mRNA1 is the only isoform encoding a full-length At-RS31 protein (9). mRNA3 and mRNA2 are almost fully retained in the nucleus ( Figure S8). mRNA1 levels decrease considerably in dark without significant changes in the total amount of At-RS31 transcripts (Figures 2A and S9) which suggests that alternative splicing is instrumental in the control of mRNA1 cellular levels and, consequently, At-RS31 protein abundance. To assess how interference with At-RS31 alternative splicing regulation could affect Arabidopsis phenotype we ...

The roles of Argonaute proteins in cytoplasmic microRNA and RNAi pathways are well established. However, their implication in small RNA-mediated transcriptional gene silencing in the mammalian cell nucleus is less understood. We have recently shown that intronic siRNAs cause chromatin modifications that inhibit RNA polymerase II elongation and modulate alternative splicing in an Argonaute-1 (AGO1)-dependent manner. Here we used chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) to investigate the genome-wide distribution of AGO1 nuclear targets. Unexpectedly, we found that about 80% of AGO1 clusters are associated with cell-type-specific transcriptional enhancers, most of them (73%) overlapping active enhancers. This association seems to be mediated by long, rather than short, enhancer RNAs and to be more prominent in intragenic, rather than intergenic, enhancers. Paradoxically, crossing ChIP-seq with RNA-seq data upon AGO1 depletion revealed that enhancer-bound AGO1 is not linked to the global regulation of gene transcription but to the control of constitutive and alternative splicing, which was confirmed by an individual gene analysis explaining how AGO1 controls inclusion levels of the cassette exon 107 in the SYNE2 gene.Argonaute proteins | transcriptional enhancers | alternative splicing A lternative splicing was initially seen as an interesting mechanism to explain protein diversity but affecting a limited number of mammalian genes. The recent development of high-throughput sequencing technologies has dramatically changed this view, generating a renewed interest in alternative splicing. We now know that alternative splicing affects transcripts from more than 90% of human genes (1) and that normal and pathological cell differentiation not only depends on differential gene expression but also on alternative splicing patterns. Mutations in alternative splicing regulatory sequences and factors are involved in the etiology of numerous hereditary diseases, premature aging, and cancer (2).Recently, amid an avalanche of papers reporting various connections between the chromatin context and splicing (3-9), a relationship between splicing and small RNAs has emerged. The convergence of these previously unrelated areas (RNA interference, chromatin, and splicing) has been studied by our laboratory, showing that siRNAs (20-25 nt long) targeting both intronic and exonic regions near the cassette exon 33 (E33, also known as EDI) of the fibronectin gene were able to regulate its alternative splicing by affecting the chromatin context at the target region, with an increase of histone tail modifications associated with gene silencing (H3K9me2 and H3K27me3, i.e., dimethylation of lysine 9 and trimethylation of lysine 27 of histone H3 respectively). Moreover, this effect was shown to be dependent on Argonaute proteins (AGO1 and AGO2) and involves a decrease of RNA polymerase II (RNAPII) elongation, which concomitantly up-regulates E33 inclusion into the mature mRNA (3). More recently, a similar effect was fou...

Alternative splicing plays a key role in generating protein diversity. Transfections with minigenes revealed coordination between two distant, alternatively spliced exons in the same gene. Mutations that either inhibit or stimulate inclusion of the upstream alternative exon deeply affect inclusion of the downstream one. However, similar mutations at the downstream alternative exon have little effect on the upstream one. This polar effect is promoter specific and is enhanced by inhibition of transcriptional elongation. Consistently, cells from mutant mice with either constitutive or null inclusion of a fibronectin alternative exon revealed coordination with a second alternative splicing region, located far downstream. Using allele-specific RT-PCR, we demonstrate that this coordination occurs in cis and is also affected by transcriptional elongation rates. Bioinformatics supports the generality of these findings, indicating that 25% of human genes contain multiple alternative splicing regions and identifying several genes with nonrandom distribution of mRNA isoforms at two alternative regions.

Alternative splicing contributes to cell type-specific transcriptomes. Here, we show that changes in intragenic chromatin marks affect NCAM (neural cell adhesion molecule) exon 18 (E18) alternative splicing during neuronal differentiation. An increase in the repressive marks H3K9me2 and H3K27me3 along the gene body correlated with inhibition of polymerase II elongation in the E18 region, but without significantly affecting total mRNA levels. Treatment with the general DNA methylation inhibitor 5-azacytidine and BIX 01294, a specific inhibitor of H3K9 dimethylation, inhibited the differentiation-induced E18 inclusion, pointing to a role for repressive marks in sustaining NCAM splicing patterns typical of mature neurons. We demonstrate that intragenic deployment of repressive chromatin marks, induced by intronic small interfering RNAs targeting NCAM intron 18, promotes E18 inclusion in undifferentiated N2a cells, confirming the chromatin changes observed upon differentiation to be sufficient to induce alternative splicing. Combined with previous evidence that neuronal depolarization causes H3K9 acetylation and subsequent E18 skipping, our results show how two alternative epigenetic marks regulate NCAM alternative splicing and E18 levels in different cellular contexts.

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