Roles of hnRNP A1, SR Proteins, and p68 Helicase in c-H-ras Alternative Splicing Regulation

Abstract

Human ras genes play central roles in coupling extracellular signals with complex intracellular networks controlling proliferation, differentiation, and apoptosis, among others processes. c-H-ras pre-mRNA can be alternatively processed into two mRNAs due to the inclusion or exclusion of the alternative exon IDX; this renders two proteins, p21H-Ras and p19H-RasIDX, which differ only at the carboxy terminus. Here, we have characterized some of the cis-acting sequences and trans-acting factors regulating IDX splicing. A downstream intronic silencer sequence (rasISS1), acting in concert with IDX, negatively regulates upstream intron splicing. This effect is mediated, at least in part, by the binding of hnRNP A1. Depletion and add-back experiments in nuclear extracts have confirmed hnRNP A1's inhibitory role in IDX splicing. Moreover, the addition of two SR proteins, SC35 and SRp40, can counteract this inhibition by strongly promoting the splicing of the upstream intron both in vivo and in vitro. Further, the RNA-dependent helicase p68 is also associated with both IDX and rasISS1 RNA, and suppression of p68 expression in HeLa cells by RNAi experiments results in a marked increase of IDX inclusion in the endogenous mRNA, suggesting a role for this protein in alternative splicing regulation.

ACKNOWLEDGMENTS

This work was supported by Fundación Ramón Areces and MCyT grants PB98-0642 and BMC2002-03282, but it was started with a grant from the Asociación Española contra el Cáncer and La Marató de TV3. This work was supported by funds from the CNRS, the INSERM, and the Association pour la Recherche sur le Cancer. S.G. was a recipient of a BEFI fellowship.

We thank the staff of the IGBMC facilities for their assistance. We thank Ruth Willmott for revising the manuscript.