DIRECT ANALYSIS OF NEMATODE CIS-SPLICEOSOMES AND TRANS-SPLICEOSOMES -A FUNCTIONAL-ROLE FOR U5 SNRNA IN SPLICED LEADER ADDITION TRANSSPLICING AND THE IDENTIFICATION OF NOVEL SM SNRNPS

Most nuclear pre-mRNAs in nematodes are processed by both cis- and tra ns-splicing. In trans-splicing, the 5' terminal exon, the spliced lead er sequence (SL), is derived from a trans-splicing specific Sm snRNP, the SL RNP, Because U snRNPs are required cofactors for trans-splicing , and because this processing reaction proceeds via a two-step reactio n pathway identical to that of cis-splicing, it has long been assumed that transsplicing is catalyzed in a complex analogous to the cis-spli ceosome. However, similarities or differences between cis- and trans-s pliceosomes have not been established. In particular, the role of U5 s nRNP in trans-splicing has been unclear. Here, we have used affinity s election to analyze the U snRNA constituents of nematode cis- and tran s-spliceosomes. We find that U5 snRNP is an integral component of the trans-spliceosome and, using site-specific crosslinking, we show that U5 snRNP establishes specific interactions with the SL RNA exon. We al so identify two novel Sm snRNPs that are enriched in both cis- and tra ns-spliceosomes. Finally, we provide evidence that a SL RNP-containing multi-snRNP (SL, U4, U5, and U6 RNPs) may be a functional precursor i n trans-spliceosome assembly.