Phosphorylation of the ASF/SF2 RS domain affects both protein-protein and protein-RNA interactions and is necessary for splicing

• Published in: Genes & development Vol. 11; no. 3; pp. 334 - 344
• Main Authors: Xiao, S H, Manley, J L
• Format: Journal Article
• Language: English
• Published: United States 01.02.1997
• Subjects: Arginine - metabolism; Binding Sites; Escherichia coli; Ions; Nuclear Proteins - metabolism; Phosphorylation; Protein Binding; Protein-Serine-Threonine Kinases - metabolism; Protein-Tyrosine Kinases - metabolism; Recombinant Proteins - metabolism; Ribonucleoprotein, U1 Small Nuclear - metabolism; RNA - metabolism; RNA Splicing; RNA-Binding Proteins - metabolism; Serine - metabolism; Serine-Arginine Splicing Factors
• ISSN: 0890-9369; 1549-5477
• DOI: 10.1101/gad.11.3.334

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.