PHOSPHORYLATION OF THE ASE SF2 RS DOMAIN AFFECTS BOTH PROTEIN-PROTEINAND 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 vitr o, contain one or two amino-terminal RNP-type RNA-binding domains and an extensively phosphorylated carboxy-terminal region enriched in repe ating Arg-Ser dipeptides (RS domains). Previous studies have suggested that RS domains participate in protein-protein interactions with othe r RS domain-containing proteins. Here we provide evidence that the RS domain of unphosphorylated recombinant ASF/SF2 is necessary, but not s ufficient, for binding to the UI 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 nonspecif ic bridging between the basic RS domain, RNA, and 70K. In vitro phosph orylation of recombinant ASF/SF2 both significantly enhanced binding t o 70K and also eliminated the RS domain-RNA interaction. Providing evi dence that these interactions are relevant to splicing, ASF/SF2 can bi nd 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 v itro.