THE FINAL STAGES OF SPLICEOSOME MATURATION REQUIRE SPP2P THAT CAN INTERACT WITH THE DEAH BOX PROTEIN PRP2P AND PROMOTE STEP-1 OF SPLICING

Pre-mRNA processing occurs by assembly of splicing factors on the subs trate to form the spliceosome followed by two consecutive RNA cleavage -ligation reactions. The Prp2 protein hydrolyzes ATP and is required f or the first reaction (Yean SL, Lin RJ, 1991, Mol Cell Biol 11:5571-55 77; Kim SH, Smith J, Claude A, Lin RJ, 1992, EMBO J11:2319-2326). The Saccharomyces cerevisiae SPPS gene was previously identified as a high -copy suppressor of temperature-sensitive prp2 mutants (Last RL, Maddo ck JR, Woolford JL Jr, 1987, Genetics 117:619-631). We have characteri zed the function of Spp2p in vivo and in vitro, Spp2p is an essential protein required for the first RNA cleavage reaction in vivo. Depletio n of Spp2p from yeast cells results in accumulation of unspliced pre-m RNAs. A temperature-sensitive spp2-1 mutant accumulates pre-mRNAs in v ivo and is unable to undergo the first splicing reaction in vitro. How ever, spliceosomal complexes are assembled in extracts prepared from t he mutant. We show that Spp2p function is required after spliceosome a ssembly but prior to the first reaction, Spp2p associates with the spl iceosome before the first RNA cleavage reaction and is likely to be re leased from the spliceosome following ATP hydrolysis by Prp2p. The Prp 2 and Spp2 proteins are capable of physically interacting with each ot her. These results suggest that Spp2p interacts with Prp2p in the spli ceosome prior to the first cleavage-ligation reaction. Spp2p is the fi rst protein that has been found to interact with a DEAD/H box splicing factor.