Splicing Function of Mammalian U6 Small Nuclear RNA: Conserved Positions in Central Domain and Helix I are Essential During the First and Second Step of Pre-mRNA Splicing

• Published in: Proceedings of the National Academy of Sciences - PNAS 1994-02, Vol.91 (3), p.903-907
• Main Authors: Wolff, Thorsten, Menssen, Ruth, Hammel, Jurgen, Bindereif, Albrecht
• Format: Article
• Language: English
• Subjects: Base Sequence; Biochemistry; Biological and medical sciences; Complementation; Conserved Sequence; function; Fundamental and applied biological sciences. Psychology; Genetic Complementation Test; Genetic mutation; HeLa Cells; Humans; Mammals; man; Models, Molecular; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; mRNA; Mutagenesis, Site-Directed; mutation; Nucleic Acid Conformation; Nucleotides; Point mutation; precursors; Ribonucleic acid; RNA; RNA Precursors - genetics; RNA Precursors - metabolism; RNA Splicing - genetics; RNA, Small Nuclear - chemistry; RNA, Small Nuclear - genetics; RNA, Small Nuclear - metabolism; Small nuclear ribonucleoproteins; Small nuclear RNA; snRNA U6; Spliceosomes; Spliceosomes - metabolism; Splicing; Transcription. Transcription factor. Splicing. Rna processing; Yeasts
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.91.3.903

On the basis of mutational analyses in yeast, the highly conserved ACAGAGA sequence of U6 small nuclear RNA (snRNA) and the adjacent U6-U2 helix I have been proposed to be part of the active center of the spliceosome. We report here a detailed analysis of the human U6 snRNA sequence requirements during the first and second step of splicing, using a mammalian in vitro splicing-complementation system and a mutational approach. Positions A53G54C55(helix Ib) were identified as important specifically for the first step, but not for spliceosome assembly. A45of the ACAGAGA sequence and U52of helix Ia function during the second step; in addition, the bulge separating helices Ia and Ib appears critical for the second step. In contrast, no splicing-essential sequences could be identified in the central domain upstream of the ACAGAGA sequence. In sum, our data demonstrate for the mammalian splicing system that discrete positions within the ACAGAGA sequence and helix I of U6 snRNA function during the first and second step of splicing, suggesting that these two sequence elements are closely associated with the catalytic center of the spliceosome. Comparison with previous results in yeast indicates a fundamental conservation of the U6 snRNA function in the pre-mRNA splicing mechanism.