Multiple properties of the splicing repressor SRp38 distinguish it from typical SR proteins.
The SR protein SRp38 is a general splicing repressor that is activated by dephosphorylation during mitosis and in response to heat shock. Here we describe experiments that provide insights into the mechanism by which SRp38 functions in splicing repression. We first show that SRp38 redistributes and colocalizes with snRNPs, but not with a typical SR protein, SC35, during mitosis and following heat shock. Supporting the functional significance of this association, a micrococcal nuclease-sensitive component, i.e., an snRNP(s), completely rescued heat shock-induced splicing repression in vitro, and purified U1 snRNP did so partially. SRp38 contains an N-terminal RNA binding domain (RBD) and a C-terminal RS domain composed of two subdomains (RS1 and RS2 domains). Unexpectedly, an RS1 deletion mutant derivative specifically inhibited the second step of splicing, while an RS2 deletion mutant retained significant dephosphorylation-dependent repression activity. Using chimeric SRp38/SC35 proteins, we show that SC35-RBD/SRp38-RS can function as a general splicing activator and that the dephosphorylated version can act as a strong splicing repressor. SRp38-RBD/SC35-RS, however, was essentially inactive in these assays. Together, our results help to define the unusual features of SRp38 that distinguish it from other SR proteins.
Shin
Chanseok
C
Department of Biological Sciences, Columbia University, New York, NY 10027, USA.
Kleiman
Frida E
FE
Manley
James L
JL
D000595:Amino Acid Sequence; D018797:Cell Cycle Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D003593:Cytoplasm / Q000737:chemistry; D006358:Hot Temperature; D006801:Humans; D008938:Mitosis; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D017434:Protein Structure, Tertiary / Q000235:genetics; D012326:RNA Splicing; D016601:RNA-Binding Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000032:analysis / Q000378:metabolism; D012261:Ribonucleoproteins / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D000068103:Serine-Arginine Splicing Factors
D018797:Cell Cycle Proteins; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D017412:Ribonucleoprotein, U1 Small Nuclear; D012261:Ribonucleoproteins; C115188:SRSF10 protein, human; C073288:SRSF2 protein, human; D000068103:Serine-Arginine Splicing Factors