MULTIPLE REQUIREMENTS FOR NEMATODE SPLICED LEADER RNP FUNCTION IN TRANSSPLICING

The 5' exon donor in nematode trans-splicing, the SL RNA, is a small ( similar to 100 nt) RNA that resembles cis-spliceosomal U snRNAs. Exten sive analyses of the RNA sequence requirements for SL RNA function hav e revealed four essential elements, the core Sm binding site, three nu cleotides immediately downstream of this site, a region of Stem-loop I I, and a 5' splice site. Although these elements are necessary and suf ficient for SL RNA function in vitro, their respective roles in promot ing SL RNA activity have not been elucidated. Furthermore, although it has been shown that assembly of the SL RNA into an Sm RNP is a prereq uisite for function, the protein composition of the SL RNP has not bee n determined. Here, we have used oligoribonucleotide affinity to purif y the SL RNP and find that it contains core Sm proteins as well as fou r specific proteins (175, 40, 30, and 28 kDa). Using in vitro assembly assays, we show that association of the 175- and 30-kDa SL-specific p roteins correlates with SL RNP function in trans-splicing. Binding of these proteins depends upon the sequence of the core Sm binding site; SL RNAs containing the U1 snRNA Sm binding site assemble into Sm RNPs that contain core, but not SL-specific proteins. Furthermore, mutation al and thiophosphate interference approaches reveal that both the prim ary nucleotide sequence and a specific phosphate oxygen within a segme nt of Stem-loop II of the SL RNA are required for function. Finally, m utational activation of an unusual cryptic 5' splice site within the S L sequence itself suggests that U5 snRNA may play a primary role in se lecting and specifying the 5' splice site in SL addition trans-splicin g.