THE HUMAN SPLICING FACTORS ASF SF2 AND SC35 POSSESS DISTINCT, FUNCTIONALLY SIGNIFICANT RNA-BINDING SPECIFICITIES/

ASF/SF2 and SC35 belong to a highly conserved family of nuclear protei ns that are both essential for splicing of pre-mRNA in vitro and are a ble to influence selection of alternative splice sites. An important q uestion is whether these proteins display distinct RNA binding specifi cities and, if so, whether this influences their functional interactio ns with pre-mRNA. To address these issues, we first performed selectio n/amplification from pools of random RNA sequences (SELEX) with portio ns of the two proteins comprising the RNA binding domains (RBDs). Alth ough both molecules selected mainly purine-rich sequences, comparison of individual sequences indicated that the motifs recognized are diffe rent. Binding assays performed with the full-length proteins confirmed that ASF/SF2 and SC35 indeed have distinct specificities, and at the same time provided evidence that the highly charged arginine-serine re gion of each protein is not a major determinant of specificity. In the case of ASF/SF2, evidence is presented that binding specificity invol ves cooperation between the protein's two RBDs. Finally, we demonstrat e that an element containing three copies of a high-affinity ASF/SF2 b inding site constitutes a powerful splicing enhancer. In contrast, a s imilar element consisting of three SC35 sites was inactive. The ASF/SF 2 enhancer can be activated specifically in splicing-deficient S100 ex tracts by recombinant ASF/SF2 in conjunction with one or more addition al protein factors. These and other results suggest a central role for ASF/SF2 in the function of purine-rich splicing enhancers.