Control of alternative splicing through siRNA-mediated transcriptional gene silencing

Abstract

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.

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

“…The existence of specific nuclear roles for these proteins in mammalian cells was demonstrated at the individual gene level for the mechanism of transcriptional gene silencing either acting at the promoter level (18,34,35) or intragenically in the regulation of alternative splicing (3,10,23). In particular, the fact that AGO1 was shown to interact with chromatin-embedded proteins such as chromatin modifiers, splicing factors (10), and RNA polymerase II (13) prompted us to define the DNA targets of AGO1 at a genomewide level using ChIP-seq in human cells.…”

“…5 B and C). All these features prompted us to investigate whether SYNE2 E107 inclusion is regulated in an AGO1-dependent manner, in particular, through the TGS-AS mechanism (3,23). Because an essential part of the TGS-AS mechanism is inhibition of RNAPII elongation, we first evaluated how SYNE2 E107 responds to activators and inhibitors of elongation.…”

“…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 found over the variant region of the endogenous CD44 gene, where both AGO1 and AGO2 were recruited after treatment with phorbol-12-myristate-13-acetate in a Dicer-dependent manner, favoring the methylation of H3K9 and the concomitant recruitment of the Significance Argonaute proteins are well characterized factors in posttranscriptional gene silencing, the process by which small RNAs trigger mRNA degradation or inhibit translation in the cytoplasm.…”

“…Recently, amid an avalanche of papers reporting various connections between the chromatin context and splicing (3)(4)(5)(6)(7)(8)(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).…”

CTCF: from insulators to alternative splicing regulation

“…Consistently, the transcription-associated H3K36me3 modification is less prominently enriched in alternatively spliced exons than in constitutive exons [8]. In an opposite way, chromatin remodelers such as SWI/SNF [9] or siRNAs that trigger silencing histone marks such as H3K9me2 and H3K27me3 through a argonaute-1-mediated transcriptional gene silencing mechanism [10] were shown to create local chromatin compactions that act as roadblocks to pol II elongation, in turn affecting alternative splicing decisions. Furthermore, several genome-wide studies showed that nucleosomes are preferentially positioned in exons [11,12,13] and this positioning may act as a transient barrier to elongation that helps exon recognition at the RNA level [14,15].…”