The conserved RNA recognition motif 3 of U2 snRNA auxiliary factor (U2AF⁶⁵) is essential in vivo but dispensable for activity in vitro

Abstract

The general splicing factor U2AF⁶⁵ recognizes the polypyrimidine tract (Py tract) that precedes 3′ splice sites and has three RNA recognition motifs (RRMs). The C-terminal RRM (RRM3), which is highly conserved, has been proposed to contribute to Py-tract binding and establish protein–protein contacts with splicing factors mBBP/SF1 and SAP155. Unexpectedly, we find that the human RRM3 domain is dispensable for U2AF⁶⁵ activity in vitro. However, it has an essential function in Schizosaccharomyces pombe distinct from binding to the Py tract or to mBBP/SF1 and SAP155. First, deletion of RRM3 from the human protein has no effect on Py-tract binding. Second, RRM123 and RRM12 select similar sequences from a random pool of RNA. Third, deletion of RRM3 has no effect on the splicing activity of U2AF⁶⁵ in vitro. However, deletion of the RRM3 domain of S. pombe U2AF⁵⁹ abolishes U2AF function in vivo. In addition, certain amino acid substitutions on the four-stranded β-sheet surface of RRM3 compromise U2AF function in vivo without affecting binding to mBBP/SF1 or SAP155 in vitro. We propose that RRM3 has an unrecognized function that is possibly relevant for the splicing of only a subset of cellular introns. We discuss the implications of these observations on previous models of U2AF function.

Keywords

• RNA-binding proteins
• splicing
• Py tract
• RRM
• RNA-binding motif