Sequence-specific RNA binding by an SR protein requires RS domain phosphorylation: creation of an SRp40-specific splicing enhancer.
We showed previously that ASF/SF2, a member of the SR protein family of splicing factors, can activate a splicing enhancer element composed of high-affinity ASF/SF2 binding sites. To determine whether other SR proteins can behave similarly, we selected a high-affinity RNA-binding site (B1) for the SR protein SRp40. Strikingly, the success of this selection was completely dependent on phosphorylation of the RS domain, as unphosphorylated SRp40 failed to select specific sequences. We show that three copies of B1 function as a strong splicing enhancer, activating an intron with suboptimal splicing signals in nuclear extracts. Enhancer activity in S100 extracts (which lack SR proteins) required SRp40 and a nuclear fraction previously found to be required for ASF/SF2-dependent splicing. Importantly, enhancer activity was lost when SRp40 was replaced by ASF/SF2 or SC35, and SRp40 was the only classical SR protein found to be associated with the enhancer. Together, our results indicate that phosphorylation-dependent, sequence-specific RNA binding can impart unique activities to individual SR proteins.
Tacke
R
R
Department of Biological Sciences, Columbia University, New York, NY 10027, USA.
Chen
Y
Y
Manley
J L
JL
D001665:Binding Sites; D002467:Cell Nucleus / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011487:Protein Conformation; D012326:RNA Splicing; D016601:RNA-Binding Proteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D000068103:Serine-Arginine Splicing Factors
D009687:Nuclear Proteins; D010750:Phosphoproteins; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D000068103:Serine-Arginine Splicing Factors