Multiple roles for U6 snRNA in the splicing pathway

• Published in: Genes & development Vol. 4; no. 12B; pp. 2264 - 2277
• Main Authors: MADHANI, H. D, BORDONNE, R, GUTHRIE, C
• Format: Journal Article
• Language: English
• Published: Cold Spring Harbor, NY Cold Spring Harbor Laboratory 01-12-1990
• Subjects: Base Sequence; Biological and medical sciences; Blotting, Northern; Fundamental and applied biological sciences. Psychology; Gene Library; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation; Nucleic Acid Conformation; Phenotype; Ribonucleoproteins - genetics; Ribonucleoproteins, Small Nuclear; RNA Splicing; RNA, Fungal - genetics; RNA, Small Nuclear - genetics; Saccharomyces cerevisiae - genetics; Transcription. Transcription factor. Splicing. Rna processing; In vitro transcription; Ribonucleoprotein; Yeast; Splicing; Site directed mutagenesis; Molecular interaction; Fungi; Northern blotting; sn-RNA; Messenger RNA; Gene; Structure activity relation; Point mutation; Ascomycetes; Molecular cloning; Saccharomyces cerevisiae; Thallophyta
• ISSN: 0890-9369; 1549-5477
• DOI: 10.1101/gad.4.12b.2264

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.