FUNCTIONAL DOMAINS OF THE HUMAN SPLICING FACTOR ASF SF2/

The human splicing factor ASF/SF2 displays two predominant activities in in vitro splicing assays: (i) it is an essential factor apparently required for all splices and (ii) it is able to switch utilization of alternative 5' splice sites in a concentration-dependent manner. ASF/S F2 is the prototype of a family of proteins typified by the presence o f one or two RNP-type RNA binding domains (RBDs) and a region highly e nriched in repeating arginine-serine dipeptides (RS regions). Here we describe a functional analysis of ASF/SF2, which defines several regio ns essential for one, or both, of its two principal activities, and pr ovides insights into how this type of protein functions in splicing. T wo isoforms of the protein, which arise from alternative splicing, are by themselves inactive, but each can block the activity of ASF/SF2, t hereby functioning as splicing repressors. Some, but not all, mutation s in the RS region prevent ASF/SF2 from functioning as an essential sp licing factor. However, the entire RS region can be deleted without re ducing splice site switching activity, indicating that it is not absol utely required for interaction with other splicing factors. Experiment s with deletion and substitution mutants reveal that the protein conta ins two related, but highly diverged, RBDs, and that both are essentia l for activity. Each RBD by itself retains the ability to bind RNA, al though optimal binding requires both domains.