FUNCTIONAL RECONSTITUTION OF U6 SNRNA IN NEMATODE CIS-SPLICING AND TRANSSPLICING - U6 CAN SERVE AS BOTH A BRANCH ACCEPTOR AND A 5'-EXON

Maturation of nuclear pre-mRNAs in nematodes requires both cis- and tr ans-splicing. Both processing pathways involve analogous two-step phos photransfer reactions and both are dependent upon the integrity of U6 snRNA. We have developed a functional reconstitution assay to assess t he U6 snRNA sequence requirements for cis- and trans-splicing. Branch formation between the splicing substrates and U6 snRNA was observed. T he frequency of this event was greatly enhanced when a highly conserve d sequence in U6 snRNA was altered by mutation. In cis- and trans-spli cing reactions reconstituted with this mutant U6 snRNA the liberated e xon of U6 proceeded through the second step of splicing using the appr opriate splice acceptor sites. These results demonstrate covalent inte ractions between a U snRNA required for splicing and a splicing substr ate, and they provide evidence for an unexpected degree of catalytic f lexibility within the spliceosome.