M(3)G CAP HYPERMETHYLATION OF U1 SMALL NUCLEAR RIBONUCLEOPROTEIN (SNRNP) IN-VITRO - EVIDENCE THAT THE U1 SMALL NUCLEAR RNA-(GUANOSINE-N2)-METHYLTRANSFERASE IS A NON-SNRNP CYTOPLASMIC PROTEIN THAT REQUIRES A BINDING-SITE ON THE SM CORE DOMAIN

The RNA components of small nuclear ribonucleoproteins (U snRNPs) poss ess a characteristic 5'-terminal trimethylguanosine cap structure (m(3 )G cap). This cap is an important component of the nuclear localizatio n signal of U snRNPs. It arises by hypermethylation of a cotranscripti onally added m(7)G cap. Here we describe an in vitro assay for the hyp ermethylation, which employs U snRNP particles reconstituted in vitro from purified components and subsequent analysis by m(3)G cap-specific immunoprecipitation. Complementation studies in vitro revealed that b oth cytosol and S-adenosylmethionine are required for the hypermethyla tion of an m(7)G-capped U1 snRNP reconstituted in vitro, indicating th at the U1 snRNA-(guanosine-N2)-methyltransferase is a trans-active non -snRNP protein. Chemical modification revealed one cytoplasmic compone nt required for hypermethylation and one located on the snRNP: these c omponents have different patterns of sensitivity to modification by N- ethylmaleimide and iodoacetic acid (IAA). In the presence of cytosol a nd S-adenosylmethionine, an intact Sm core domain is a necessary and s ufficient substrate for cap hypermethylation. These data, together wit h our observation that isolated native U1 snRNPs but not naked U1 RNA inhibit the trimethylation of in vitro-reconstituted U1 snRNP, indicat e that the Sm core binds the methyltransferase specifically. Moreover, isolated native U2 snRNP also inhibits trimethylation of U1 snRNP, su ggesting that other Sm-class U snRNPs might share the same methyltrans ferase. IAA modification of m(7)G-capped U1 snRNPs inhibited hypermeth ylation when they were microinjected into Xenopus oocytes and conseque ntly also inhibited nuclear import. In contrast, modification with IAA of m(3)G-capped U1 snRNPs reconstituted in vitro did not interfere wi th their nuclear transport in oocytes. These data suggest that m(3)G c ap formation and nuclear transport of U1 snRNPs are mediated by distin ct factors, which require distinct binding sites on the Sm core of U1 snRNP.