Control of alternative splicing through siRNA-mediated transcriptional gene silencing

• Published in: Nature structural & molecular biology Vol. 16; no. 7; pp. 717 - 724
• Main Authors: Alló, Mariano, Buggiano, Valeria, Fededa, Juan P, Petrillo, Ezequiel, Schor, Ignacio, de la Mata, Manuel, Agirre, Eneritz, Plass, Mireya, Eyras, Eduardo, Elela, Sherif Abou, Klinck, Roscoe, Chabot, Benoit, Kornblihtt, Alberto R
• Format: Journal Article
• Language: English
• Published: New York Nature Publishing Group US 01.07.2009; Nature Publishing Group
• Subjects: Alternative Splicing; Animals; Argonaute Proteins; Base Sequence; Biochemistry; Biological Microscopy; Biomedical and Life Sciences; Carcinoma, Hepatocellular - genetics; Carcinoma, Hepatocellular - metabolism; Chromatin; Epigenesis, Genetic; Eukaryotic Initiation Factors; Exons; Fibronectins - genetics; Fibronectins - metabolism; Gene Knockdown Techniques; Genetic engineering; Genetic transcription; Genetics; HeLa Cells; Heterochromatin - genetics; Heterochromatin - metabolism; Histones - genetics; Histones - metabolism; Humans; Hybridization; Kinetics; Life Sciences; Liver Neoplasms - genetics; Liver Neoplasms - metabolism; Lysine - metabolism; Male; Membrane Biology; Methylation; Mice; Oligonucleotides, Antisense - genetics; Oligonucleotides, Antisense - metabolism; Physiological aspects; Promoter Regions, Genetic; Protein Structure; Ribonuclease III - genetics; Ribonuclease III - metabolism; Ribonucleic acid; RNA; RNA Interference; RNA Polymerase II - genetics; RNA Polymerase II - metabolism; RNA, Small Interfering - genetics; RNA, Small Interfering - metabolism; Transcription, Genetic; Argentina; Spain
• ISSN: 1545-9993; 1545-9985; 1545-9985
• DOI: 10.1038/nsmb.1620

Exogenously applied small RNAs have previously been shown to inhibit transcriptional levels when targeted to promoters. They are now shown to alter the ratio of alternative splice forms. The features of splice form alteration are reminiscent of transcriptional gene silencing by siRNAs. 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 HP1α and the reduction in RNA polymerase II processivity suggest a mechanism involving the kinetic coupling of transcription and alternative splicing.