P68 RNA Helicase (DDX5) Alters Activity of Cis- and Trans-Acting Factors of the Alternative Splicing of H-Ras

Maria Camats,Sonia Guil,Mariette Kokolo,Montse Bach-Elias,Juan Valcarcel

doi:10.1371/journal.pone.0002926

Maria Camats, Sonia Guil + Show 3 more

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https://doi.org/10.1371/journal.pone.0002926

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Abstract

BackgroundH-Ras pre-mRNA undergoes an alternative splicing process to render two proteins, namely p21 H-Ras and p19 H-Ras, due to either the exclusion or inclusion of the alternative intron D exon (IDX), respectively. p68 RNA helicase (p68) is known to reduce IDX inclusion.Principal FindingsHere we show that p68 unwinds the stem-loop IDX-rasISS1 structure and prevents binding of hnRNP H to IDX-rasISS1. We also found that p68 alters the dynamic localization of SC35, a splicing factor that promotes IDX inclusion. The knockdown of hnRNP A1, FUS/TLS and hnRNP H resulted in upregulation of the expression of the gene encoding the SC35-binding protein, SFRS2IP. Finally, FUS/TLS was observed to upregulate p19 expression and to stimulate IDX inclusion, and in vivo RNAi-mediated depletion of hnRNP H decreased p19 H-Ras abundance.SignificanceTaken together, p68 is shown to be an essential player in the regulation of H-Ras expression as well as in a vital transduction signal pathway tied to cell proliferation and many cancer processes.

Highlights

H, K- and N-Ras proteins play a significant role in controlling cell growth, a central component of mitogenic signal transduction pathways, reviewed in [1,2]
Studies using RNA affinity columns containing either intron D exon (IDX), rasISS1 or the mutant DrasISS1 in presence of nuclear extract showed that p68, hnRNP H and FUS bound to IDX and rasISS1, but not to DrasISS1 [8]
Previous in vivo studies found that RNAi-mediated depletion of p68 increased the level of endogenous p19 mature mRNA, thereby revealing a role for p68 RNA helicase as an inhibitor of IDX inclusion [8]

Summary

Introduction

H-, K- and N-Ras proteins play a significant role in controlling cell growth, a central component of mitogenic signal transduction pathways, reviewed in [1,2]. Using winding/unwinding assays, we analyzed how p68 affects IDXrasISS1 stem-loop structure and the binding of hnRNP H and FUS/TLS to this IDX-rasISS1 region.

Results

Conclusion