SERINE PHOSPHORYLATION OF SR PROTEINS IS REQUIRED FOR THEIR RECRUITMENT TO SITES OF TRANSCRIPTION IN-VIVO

Expression of most RNA polymerase II transcripts requires the coordina ted execution of transcription, splicing, and 3' processing. We have p reviously shown that upon transcriptional activation of a gene in vivo , pre-mRNA splicing factors are recruited from nuclear speckles, in wh ich they are concentrated, to sites of transcription (Misteli, T., J.F . Caceres, and D.L. Spector. 1997. Nature. 387:523-527). This recruitm ent process appears to spatially coordinate transcription and pre-mRNA splicing within the cell nucleus. Here we have investigated the molec ular basis for recruitment by analyzing the recruitment properties of mutant splicing factors. We show that multiple protein domains are req uired for efficient recruitment of SR proteins from nuclear speckles t o nascent RNA. The two types of modular domains found in the splicing factor SF2/ ASF exert distinct functions in this process. In living ce lls, the RS domain functions in the dissociation of the protein from s peckles, and phosphorylation of serine residues in the RS domain is a prerequisite for this event. The RNA binding domains play a role in th e association of splicing factors with the target RNA. These observati ons identify a novel in vivo role for the RS domain of SR proteins and suggest a model in which protein phosphorylation is instrumental for the recruitment of these proteins to active sites of transcription in vivo.