Multiple roles for U6 snRNA in the splicing pathway.
U6 is the most highly conserved of the five spliceosomal RNAs. It is associated with U4 by an extensive base-pairing interaction, which is disrupted immediately prior to the first nucleolytic step of splicing. It has been proposed that this event activates catalysis by unmasking U6. Using a combination of doped synthesis and site-directed mutagenesis to generate point mutations in U6, we have now identified 12 positions, in three domains, at which single nucleotide substitutions or deletions result in lethal or temperature-sensitive phenotypes. Biochemical analysis demonstrates that most of these mutants retain the ability to assemble into U4/U6 and U4/U5/U6 snRNPs. Notably, although mutations at three positions in U6 that base-pair with U4 are lethal, mutations in the complementary residues in U4 are fully viable. Furthermore, compensatory mutations in U4 that restore base-pairing fail to suppress the phenotypes of the U6 mutations. This demonstrates a function for U6 independent of its role in base-pairing. Remarkably, two of the three essential regions in U6 identified genetically correspond to intron insertion points in two yeast species. A temperature-sensitive mutation at one of these sites is defective in the second step of splicing in vitro.
Madhani
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HD
Department of Biochemistry and Biophysics, University of California, San Francisco 94143.
Bordonné
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Guthrie
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D001483:Base Sequence; D015152:Blotting, Northern; D015723:Gene Library; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009690:Nucleic Acid Conformation; D010641:Phenotype; D012326:RNA Splicing; D012331:RNA, Fungal / Q000235:genetics; D012342:RNA, Small Nuclear / Q000235:genetics*; D012261:Ribonucleoproteins / Q000235:genetics; D017411:Ribonucleoproteins, Small Nuclear; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D012331:RNA, Fungal; D012342:RNA, Small Nuclear; D012261:Ribonucleoproteins; D017411:Ribonucleoproteins, Small Nuclear