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 HP1α and the reduction in RNA polymerase II processivity suggest a mechanism involving the kinetic coupling of transcription and alternative splicing.
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Acknowledgements
We thank P. Bertucci, M. Blaustein, F. Pelisch, M. Muñoz, A. Srebrow, G. Risso, L.G. Acuña, M.G. Herz, N. Tilgner, I. Listerman, M. Bühler, J. Martínez, K. Neugebauer and K. Morris for their support and useful discussions; and Boehringer Ingelheim Pharmaceuticals (Biomolecular Screening, Department of Medicinal Chemistry) for the gift of BIX-01294. This work was supported by grants to A.R.K. from the Fundación Antorchas, the Agencia Nacional de Promoción de Ciencia y Tecnología of Argentina, the University of Buenos Aires and the European Alternative Splicing Network. S.A.E., R.K. and B.C. acknowledge support from Genome Canada and Genome Quebec. M.A. is the recipient of a fellowship and A.R.K. is a career investigator from the Consejo Nacional de Investigaciones Científicas y Técnicas of Argentina. B.C. holds a Canada Research Chair in functional genomics. A.R.K. is an international research scholar of the Howard Hughes Medical Institute.
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M.A. proposed, designed and conducted most of the experiments and prepared the manuscript; V.B., J.P.F., E.P., I.S. and M.d.l.M. conducted some experiments; E.A., M.P. and E.E. conducted the bioinformatics analysis; S.A.E., R.K. and B.C. conducted the PCR panel experiment; and A.R.K. coordinated the work and prepared the manuscript.
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Alló, M., Buggiano, V., Fededa, J. et al. Control of alternative splicing through siRNA-mediated transcriptional gene silencing. Nat Struct Mol Biol 16, 717–724 (2009). https://doi.org/10.1038/nsmb.1620
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DOI: https://doi.org/10.1038/nsmb.1620
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