Roles of hnRNP A1, SR proteins, and p68 helicase in c-H-ras alternative splicing regulation.
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.
Guil
Sònia
S
Institut d'Investigació Cardiovascular de Barcelona-Consejo Superior de Investigaciones Científicas, Barcelona, Spain.
Gattoni
Renata
R
Carrascal
Montserrat
M
Abián
Joaquín
J
Stévenin
James
J
Bach-Elias
Montse
M
D017398:Alternative Splicing; D001483:Base Sequence; D053487:DEAD-box RNA Helicases; D011905:Genes, ras; D006367:HeLa Cells; D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D010750:Phosphoproteins / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D011494:Protein Kinases / Q000378:metabolism*; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics / Q000378:metabolism; D020365:RNA Helicases / Q000378:metabolism*; D034622:RNA Interference; D012322:RNA Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012261:Ribonucleoproteins; D000068103:Serine-Arginine Splicing Factors; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism
D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D009687:Nuclear Proteins; D010750:Phosphoproteins; D012322:RNA Precursors; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D012261:Ribonucleoproteins; C073288:SRSF2 protein, human; D000068103:Serine-Arginine Splicing Factors; D011494:Protein Kinases; C506749:Ddx5 protein, human; D053487:DEAD-box RNA Helicases; D020365:RNA Helicases; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins