Argonaute-1 binds transcriptional enhancers and controls constitutive and alternative splicing in human cells

Abstract

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...

Salt Stress Reveals a New Role for ARGONAUTE1 in miRNA Biogenesis at the Transcriptional and Posttranscriptional Levels

“…This interaction may also affect RNAPII processivity and abolish splicing of selected alternative exons (Ameyar-Zazoua et al, 2012;Li et al, 2012b;Alló et al, 2014). There have also been some suggestions that, in Arabidopsis, AGO1 localization is not limited to the cytoplasm (Ye et al, 2012;Duan et al, 2012).…”

mRNA Decay of Most Arabidopsis miRNA Targets Requires Slicer Activity of AGO1

“…Alternatively, phenotypic differences could be related to functions of AGO1 other than miRNA target repression. Indeed, the implication of AGO proteins in several pathways such as nonsense-mediated mRNA decay (Choe et al, 2010), alternative splicing (AmeyarZazoua et al, 2012;Yang et al, 2012b;Taliaferro et al, 2013;Alló et al, 2014), DNA double-strand break repair (Michalik et al, 2012;Wei et al, 2012), nucleosome occupancy at transcription start sites (Carissimi et al, 2015), and quality control of mRNAs encoding proteins entering the secretory pathway (Karamyshev et al, 2014) has been proposed during the last years. If that is the case, the conditional slicer-deficient alleles might be a valuable tool to investigate novel functions of plant AGO1 by decoupling these from target regulation.…”

RNA–RNA interactions in gene regulation: the coding and noncoding players

“…Kornblihtt and colleagues have shown that siRNAs complementary to intronic or exonic sequences close to an alternative exon can regulate the splicing of that exon by inducing facultative heterochromatin formation [30,31]. In the case of the zinc finger E-box binding homeobox 2 (Zeb2) gene (a transcriptional repressor of E-cadherin), a natural antisense transcript that overlaps the 5 0 splice site of an intron harboring an internal ribosome entry site (IRES) necessary for the expression of Zeb2 prevents its splicing [32].…”