Control of hnRNP A1 Alternative Splicing: an Intron Element Represses Use of the Common 3′ Splice Site

Abstract

Alternative splicing of exon 7B in the hnRNP A1 pre-mRNA produces mRNAs encoding two proteins: hnRNP A1 and the less abundant A1B. We have reported the identification of several intron elements that contribute to exon 7B skipping. In this study, we report the activity of a novel element, conserved element 9 (CE9), located in the intron downstream of exon 7B. We show that multiple copies of CE9 inhibit exon 7B-exon 8 splicing in vitro. When CE9 is inserted between two competing 3′ splice sites, a single copy of CE9 decreases splicing to the distal 3′ splice site. Our in vivo results also support the conclusion that CE9 is a splicing modulator. First, inserting multiple copies of CE9 into an A1 minigene compromises the production of fully spliced products. Second, one copy of CE9 stimulates the inclusion of a short internal exon in a derivative of the human β-globin gene. In this case, in vitro splicing assays suggest that CE9 decreases splicing of intron 1, an event that improves splicing of intron 2 and decreases skipping of the short internal exon. The ability of CE9 to act on heterologous substrates, combined with the results of a competition assay, suggest that the activity of CE9 is mediated by atrans-acting factor. Our results indicate that CE9 represses the use of the common 3′ splice site in the hnRNP A1 alternative splicing unit.

ACKNOWLEDGMENTS

We thank D. Black and E. Modaferri for kindly providing DUP4-1 and DUP5-1 plasmids. We thank Johanne Toutant for performing transfections and preparing nuclear extracts, and we thank M. Blanchette and S. Hutchison for comments on the manuscript.

M.J.S. is the recipient of a studentship from the FCAR/FRSQ. This work was supported by a grant from the Medical Research Council of Canada. B.C. is a Chercheur-Boursier Senior from the FRSQ and is a member of the Sherbrooke RNA/RNP group supported by the FCAR.