Phosphorylation of the ASF/SF2 RS domain affects both protein-protein and protein-RNA interactions and is necessary for splicing.
ASF/SF2 is a member of a conserved family of splicing factors known as SR proteins. These proteins, which are necessary for splicing in vitro, contain one or two amino-terminal RNP-type RNA-binding domains and an extensively phosphorylated carboxy-terminal region enriched in repeating Arg-Ser dipeptides (RS domains). Previous studies have suggested that RS domains participate in protein-protein interactions with other RS domain-containing proteins. Here we provide evidence that the RS domain of unphosphorylated recombinant ASF/SF2 is necessary, but not sufficient, for binding to the U1 snRNP-specific 70-kD protein (70K) in vitro. An apparent interaction of the isolated RS domain with 70K was observed if contaminating RNA was not removed, suggesting a nonspecific bridging between the basic RS domain, RNA, and 70K. In vitro phosphorylation of recombinant ASF/SF2 both significantly enhanced binding to 70K and also eliminated the RS domain-RNA interaction. Providing evidence that these interactions are relevant to splicing, ASF/SF2 can bind selectively to U1 snRNP in an RS domain-dependent, phosphorylation-enhanced manner. We also describe conditions that reveal for the first time a phosphorylation requirement for ASF/SF2 splicing activity in vitro.
Xiao
S H
SH
Columbia University, Department of Biological Sciences, New York, New York 10027, USA.
Manley
J L
JL
D001120:Arginine / Q000378:metabolism; D001665:Binding Sites; D004926:Escherichia coli; D007477:Ions; D009687:Nuclear Proteins / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D012313:RNA / Q000378:metabolism*; D012326:RNA Splicing; D016601:RNA-Binding Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D000068103:Serine-Arginine Splicing Factors
D007477:Ions; D009687:Nuclear Proteins; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D017412:Ribonucleoprotein, U1 Small Nuclear; C490688:SNRNP70 protein, human; D000068103:Serine-Arginine Splicing Factors; D012694:Serine; D012313:RNA; D001120:Arginine; C066780:Clk dual-specificity kinases; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases