title: Rapid screening of yeast mutants with reporters identifies new splicing phenotypes.
abstract: Nuclear precursor mRNA splicing requires the stepwise assembly of a large complex, the spliceosome. Recent large-scale analyses, including purification of splicing complexes, high-throughput genetic screens and interactomic studies, have linked numerous factors to this dynamic process, including a well-defined core conserved from yeast to human. Intriguingly, despite extensive studies, no splicing defects were reported for some of the corresponding yeast mutants. To resolve this paradox, we screened a collection of viable yeast strains carrying mutations in splicing-related factors with a set of reporters including artificial constructs carrying competing splice sites. Previous analyses have indeed demonstrated that this strategy identifies yeast factors able to regulate alternative splicing and whose properties are conserved in human cells. The method, sensitive to subtle defects, revealed new splicing phenotypes for most analyzed factors such as the Urn1 protein. Interestingly, a mutant of PRP8 specifically lacking an N-terminal proline-rich region stimulated the splicing of a reporter containing competing branchpoint/3' splice site regions. Thus, using appropriate reporters, yeast can be used to quickly delineate the effect of various factors on splicing and identify those with the propensity to regulate alternative splicing events.
authors.affiliation: Equipe Labellisée La Ligue, Institut de Génétique et de Biologie Moléculaire et Cellulaire, Université de Strasbourg, 67404 Illkirch, France.
authors.forename: Natacha
authors.initials: N
authors.lastname: Dreumont
authors.forename: Bertrand
authors.initials: B
authors.lastname: Séraphin
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D017930:Genes, Reporter; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D022821:RNA Splice Sites / Q000235:genetics*; D012326:RNA Splicing / Q000235:genetics*; D012331:RNA, Fungal / Q000235:genetics* / Q000378:metabolism; D017414:Ribonucleoprotein, U4-U6 Small Nuclear / Q000235:genetics; D017415:Ribonucleoprotein, U5 Small Nuclear / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D017381:Spliceosomes / Q000235:genetics* / Q000378:metabolism
chemical_list: C077018:PRP8 protein, S cerevisiae; D022821:RNA Splice Sites; D012331:RNA, Fungal; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; D017415:Ribonucleoprotein, U5 Small Nuclear; D029701:Saccharomyces cerevisiae Proteins