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Rapid screening of yeast mutants with reporters identifies new splicing phenotypes

Authors

  • Natacha Dreumont,

    1. Equipe Labellisée La Ligue, Institut de Génétique et de Biologie Moléculaire et Cellulaire, Université de Strasbourg, Illkirch, France
    Current affiliation:
    1. INSERM U954, Faculté de Médecine, 54505 Vandoeuvre Les Nancy Cedex, France
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  • Bertrand Séraphin

    Corresponding author
    • Equipe Labellisée La Ligue, Institut de Génétique et de Biologie Moléculaire et Cellulaire, Université de Strasbourg, Illkirch, France
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Correspondence

B. Séraphin, Equipe Labellisée La Ligue, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Institut National de Santé et de Recherche Médicale (INSERM) U964/Centre National de Recherche Scientifique (CNRS) UMR 7104/Université de Strasbourg, 67404 Illkirch, France

Fax: +33 (0)3 88 65 33 37

Tel: +33 (0)3 88 65 33 36

E-mail: seraphin@igbmc.fr

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.

Structured digital abstract

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