Control of alternative splicing through siRNA-mediated transcriptional gene silencing.
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.
Alló
Mariano
M
Laboratorio de Fisiología y Biología Molecular, Departamento de Fisiología, Biología Molecular y Celular, IFIBYNE-CONICET, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Buenos Aires, Argentina.
Buggiano
Valeria
V
Fededa
Juan P
JP
Petrillo
Ezequiel
E
Schor
Ignacio
I
de la Mata
Manuel
M
Agirre
Eneritz
E
Plass
Mireya
M
Eyras
Eduardo
E
Elela
Sherif Abou
SA
Klinck
Roscoe
R
Chabot
Benoit
B
Kornblihtt
Alberto R
AR
D017398:Alternative Splicing; D000818:Animals; D060565:Argonaute Proteins; D001483:Base Sequence; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000378:metabolism; D000090266:Chromobox Protein Homolog 5; D044127:Epigenesis, Genetic; D039642:Eukaryotic Initiation Factors; D005091:Exons; D005353:Fibronectins / Q000235:genetics / Q000378:metabolism; D055785:Gene Knockdown Techniques; D006367:HeLa Cells; D006570:Heterochromatin / Q000235:genetics / Q000378:metabolism; D006657:Histones / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics / Q000378:metabolism; D008239:Lysine / Q000378:metabolism; D008297:Male; D008745:Methylation; D051379:Mice; D016376:Oligonucleotides, Antisense / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D012319:RNA Polymerase II / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D043244:Ribonuclease III / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
C115848:AGO1 protein, human; D060565:Argonaute Proteins; C000715594:CBX5 protein, human; D039642:Eukaryotic Initiation Factors; D005353:Fibronectins; D006570:Heterochromatin; D006657:Histones; D016376:Oligonucleotides, Antisense; D034741:RNA, Small Interfering; D000090266:Chromobox Protein Homolog 5; D012319:RNA Polymerase II; D043244:Ribonuclease III; D008239:Lysine