A novel base-pairing interaction between U2 and U6 snRNAs suggests a mechanism for the catalytic activation of the spliceosome

Prior to the chemical steps of mRNA splicing, the extensive base-pairing interaction between the U4 and U6 spliceosomal snRNAs is disrupted. Here, we use a mutational analysis in yeast to demonstrate a conserved base-pairing interaction between the U6 and U2 snRNAs that is mutually exclusive with the U4-U6 interaction. In this novel pairing, conserved sequences in U6 interact with a sequence in U2 that is immediately upstream of the branch point recognition region. Remarkably, the residues in U6 that can be consequently juxtaposed with the intron substrate include those that have been proposed previously to be catalytic. Both the first and second steps of splicing are inhibited when this base-paired structure is mutated. These observations, together with the high conservation of the U2-U6 structure, lead us to propose that it might be a component of the spliceosomal active site.

[1]  S. Dib-Hajj,et al.  Group II introns deleted for multiple substructures retain self-splicing activity. , 1992, Molecular and cellular biology.

[2]  C. Guthrie.  Messenger RNA splicing in yeast: clues to why the spliceosome is a ribonucleoprotein. , 1991, Science.

[3]  M. Green,et al.  An ordered pathway of snRNP binding during mammalian pre‐mRNA splicing complex assembly. , 1987, The EMBO journal.

[4]  A. Bindereif,et al.  Reconstituted mammalian U4/U6 snRNP complements splicing: a mutational analysis. , 1992, The EMBO journal.

[5]  P Linder,et al.  D‐E‐A‐D protein family of putative RNA helicases , 1992, Molecular microbiology.

[6]  C. Norman,et al.  U5 snRNA interacts with exon sequences at 5′ and 3′ splice sites , 1992, Cell.

[7]  Y. Shimura,et al.  Association of U6 snRNA with the 5'-splice site region of pre-mRNA in the spliceosome. , 1992, Genes & development.

[8]  L. Miraglia,et al.  Limited functional equivalence of phylogenetic variation in small nuclear RNA: yeast U2 RNA with altered branchpoint complementarity inhibits splicing and produces a dominant lethal phenotype. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[9]  A. Weiner,et al.  Evidence for base-pairing between mammalian U2 and U6 small nuclear ribonucleoprotein particles. , 1990, Genes & development.

[10]  P. Sharp,et al.  Spliceosome assembly involves the binding and release of U4 small nuclear ribonucleoprotein. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[11]  S. Cheng,et al.  Spliceosome assembly in yeast. , 1987, Genes & development.

[12]  P. Sharp,et al.  "Five easy pieces". , 1991, Science.

[13]  P. Sharp,et al.  Interactions between small nuclear ribonucleoprotein particles in formation of spliceosomes , 1987, Cell.

[14]  R. Lührmann,et al.  Localization of a base-paired interaction between small nuclear RNAs U4 and U6 in intact U4/U6 ribonucleoprotein particles by psoralen cross-linking. , 1985, Journal of molecular biology.

[15]  P. Sharp,et al.  Site-specific modification of pre-mRNA: the 2'-hydroxyl groups at the splice sites. , 1992, Science.

[16]  C. Guthrie,et al.  Suppressors of a U4 snRNA mutation define a novel U6 snRNP protein with RNA-binding motifs. , 1991, Genes & development.

[17]  A. Lamond,et al.  Probing the structure and function of U2 snRNP with antisense oligonucleotides made of 2′-OMe RNA , 1989, Cell.

[18]  D. S. McPheeters,et al.  In vitro assembly of yeast U6 snRNP: a functional assay. , 1989, Genes & development.

[19]  K. Umesono,et al.  Comparative and functional anatomy of group II catalytic introns--a review. , 1989, Gene.

[20]  I. Mattaj,et al.  An abundant U6 snRNP found in germ cells and embryos of Xenopus laevis. , 1989, The EMBO journal.

[21]  T. Cech.  The generality of self-splicing RNA: Relationship to nuclear mRNA splicing , 1986, Cell.

[22]  C. Guthrie,et al.  Splicing a spliceosomal RNA , 1989, Nature.

[23]  P. Perlman,et al.  Group II intron domain 5 facilitates a trans-splicing reaction , 1988, Molecular and cellular biology.

[24]  R. Schweyen,et al.  Self-splicing of group II introns in vitro: Mapping of the branch point and mutational inhibition of lariat formation , 1986, Cell.

[25]  H. Madhani,et al.  Multiple roles for U6 snRNA in the splicing pathway. , 1990, Genes & development.

[26]  F. Michel,et al.  Multiple exon-binding sites in class II self-splicing introns , 1987, Cell.

[27]  C. Guthrie,et al.  An essential snRNA from S. cerevisiae has properties predicted for U4, including interaction with a U6-like snRNA , 1987, Cell.

[28]  Christine Guthrie,et al.  A U-rich tract enhances usage of an alternative 3′ splice site in yeast , 1991, Cell.

[29]  J. Manley,et al.  Base pairing between U2 and U6 snRNAs is necessary for splicing of a mammalian pre-mRNA , 1991, Nature.

[30]  A. Weiner,et al.  A compensatory base change in human U2 snRNA can suppress a branch site mutation. , 1989, Genes & development.

[31]  P. Fabrizio,et al.  Two domains of yeast U6 small nuclear RNA required for both steps of nuclear precursor messenger RNA splicing , 1990, Science.

[32]  R. Lin,et al.  U4 small nuclear RNA dissociates from a yeast spliceosome and does not participate in the subsequent splicing reaction , 1991, Molecular and cellular biology.

[33]  R. Lührmann,et al.  Evidence for the existence of snRNAs U4 and U6 in a single ribonucleoprotein complex and for their association by intermolecular base pairing. , 1984, The EMBO journal.

[34]  B. Séraphin,et al.  Identification of functional U1 snRNA-pre-mRNA complexes committed to spliceosome assembly and splicing , 1989, Cell.

[35]  Y. Ohshima,et al.  mRNA-type introns in U6 small nuclear RNA genes: implications for the catalysis in pre-mRNA splicing. , 1991, Genes & development.

[36]  G. Natsoulis,et al.  5-Fluoroorotic acid as a selective agent in yeast molecular genetics. , 1987, Methods in enzymology.

[37]  A. Jacquier,et al.  Splice site selection and role of the lariat in a group II intron. , 1991, Journal of molecular biology.

[38]  C. Guthrie,et al.  An essential yeast snRNA with a U5-like domain is required for splicing in vivo , 1987, Cell.

[39]  J. Steitz,et al.  U4 and U6 RNAs coexist in a single small nuclear ribonucleoprotein particle. , 1984, Nucleic acids research.

[40]  C. Guthrie,et al.  Small nuclear RNAs from budding yeasts: phylogenetic comparisons reveal extensive size variation. , 1989, Gene.

[41]  M. Green,et al.  Discrete domains of human U6 snRNA required for the assembly of U4/U6 snRNP and splicing complexes. , 1990, The EMBO journal.

[42]  C. McGuigan,et al.  Domains of U4 and U6 snRNAs required for snRNP assembly and splicing complementation in Xenopus oocytes. , 1990, The EMBO journal.

[43]  K. Köhrer,et al.  Preparation of high molecular weight RNA. , 1991, Methods in enzymology.

[44]  C. McGuigan,et al.  Roles of U4 and U6 snRNAs in the assembly of splicing complexes. , 1992, The EMBO journal.

[45]  C. Guthrie,et al.  Human U2 snRNA can function in pre-mRNA splicing in yeast , 1990, Nature.

[46]  M. Rosbash,et al.  Electrophoresis of ribonucleoproteins reveals an ordered assembly pathway of yeast splicing complexes , 1986, Nature.

[47]  M. Rosbash,et al.  Expression of a beta-galactosidase gene containing the ribosomal protein 51 intron is sensitive to the rna2 mutation of yeast. , 1983, Proceedings of the National Academy of Sciences of the United States of America.

[48]  J. Steitz,et al.  Interactions of small nuclear RNA's with precursor messenger RNA during in vitro splicing. , 1992, Science.

[49]  William A. Noon,et al.  Intron splicing: a conserved internal signal in introns of Drosophila pre-mRNAs , 1985, Nucleic Acids Res..

[50]  M. Green,et al.  Biochemical mechanisms of constitutive and regulated pre-mRNA splicing. , 1991, Annual review of cell biology.

[51]  C. Branlant,et al.  An intron in the genes for U3 small nucleolar RNAs of the yeast Saccharomyces cerevisiae. , 1990, Science.

[52]  J. Manley,et al.  Mammalian pre-mRNA branch site selection by U2 snRNP involves base pairing. , 1989, Genes & development.

[53]  Christine Guthrie,et al.  Recognition of the TACTAAC box during mRNA splicing in yeast involves base pairing to the U2-like snRNA , 1987, Cell.

[54]  A. Weiner,et al.  Genetic evidence for base pairing between U2 and U6 snRNA in mammalian mRNA splicing , 1991, Nature.

[55]  Christine Guthrie,et al.  Spliceosomal RNA U6 is remarkably conserved from yeast to mammals , 1988, Nature.

[56]  L. Guarente,et al.  Vectors for expression of cloned genes in yeast: regulation, overproduction, and underproduction. , 1991, Methods in enzymology.

[57]  C. Guthrie,et al.  Two conserved domains of yeast U2 snRNA are separated by 945 nonessential nucleotides , 1988, Cell.

[58]  Y. Ohshima,et al.  The gene for the U6 small nuclear RNA in fission yeast has an intron , 1989, Nature.

[59]  Phillip A. Sharp,et al.  On the origin of RNA splicing and introns , 1985, Cell.

[60]  R. W. Davis,et al.  A family of versatile centromeric vectors designed for use in the sectoring-shuffle mutagenesis assay in Saccharomyces cerevisiae. , 1988, Gene.

[61]  C. Norman,et al.  Mutations in yeast U5 snRNA alter the specificity of 5′ splice-site cleavage , 1991, Cell.