A HUMAN PROTEIN REQUIRED FOR THE 2ND-STEP OF PRE-MESSENGER-RNA SPLICING IS FUNCTIONALLY RELATED TO A YEAST SPLICING FACTOR

We have identified a human splicing factor required for the second ste p of pre-mRNA splicing. This new protein, hPrp18, is 30% identical to the yeast splicing factor Prp18. In HeLa cell extracts immunodepleted of hPrp18, the second step of pre-mRNA splicing is abolished. Splicing activity is restored by the addition of recombinant hPrp18, demonstra ting that hPrp18 is required for the second step. The hPrp18 protein i s bound tightly to the spliceosome only during the second step of spli cing. hPrp18 is required for the splicing of several pre-mRNAs, making it the first general second-step splicing factor found in humans. Spl icing activity can be restored to hPrp18-depleted HeLa cell extracts b y yeast Prp18, showing that important functional regions of the protei ns have been conserved. A 90-amino-acid region near the carboxyl termi nus of hPrp18 is strongly homologous to yeast Prp18 and is also conser ved in rice and nematodes. The homology identifies one region importan t for the function of both proteins and may define a new protein motif . In contrast to yeast Prp18, hPrp18 is not stably associated with any of the snRNPs. A 55-kD protein that cross-reacts with antibodies agai nst hPrp18 is a constituent of the U4/U6 and U4/U6 . U5 snRNP particle s.