A conserved Lsm-interaction motif in Prp24 required for efficient U4/U6 di-snRNP formation.
The assembly of the U4 and U6 snRNPs into the U4/U6 di-snRNP is necessary for pre-mRNA splicing, and in Saccharomyces cerevisiae requires the splicing factor Prp24. We have identified a family of Prp24 homologs that includes the human protein SART3/p110nrb, which had been identified previously as a surface antigen in several cancers. Sequence conservation among the Prp24 homologs reveals the existence of a fourth previously unidentified RNA recognition motif (RRM) in Prp24, which we demonstrate is necessary for growth of budding yeast at 37 degrees C. The family is also characterized by a highly conserved 12-amino-acid motif at the extreme C terminus. Deletion of this motif in Prp24 causes a cold-sensitive growth phenotype and a decrease in base-paired U4/U6 levels in vivo. The mutant protein also has a reduced association with U6 snRNA in extract, and is unable to interact with the U6 Lsm proteins by two-hybrid assay. In vitro annealing assays demonstrate that deletion of the motif causes a defect in U4/U6 formation by reducing binding of Prp24 to its substrate. We conclude that the conserved C-terminal motif of Prp24 interacts with the Lsm proteins to promote U4/U6 formation.
Rader
Stephen D
SD
Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, California 94143-0448, USA.
Guthrie
Christine
C
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D015152:Blotting, Northern; D016384:Consensus Sequence; D017931:DNA Primers / Q000737:chemistry; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010641:Phenotype; D016133:Polymerase Chain Reaction; D011233:Precipitin Tests; D012326:RNA Splicing; D012331:RNA, Fungal / Q000235:genetics* / Q000378:metabolism; D012342:RNA, Small Nuclear / Q000235:genetics*; D017414:Ribonucleoprotein, U4-U6 Small Nuclear / Q000235:genetics* / Q000378:metabolism; D017411:Ribonucleoproteins, Small Nuclear / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013489:Suppression, Genetic; D020798:Two-Hybrid System Techniques
D017931:DNA Primers; D005656:Fungal Proteins; C401305:Prp24 protein, S cerevisiae; D012331:RNA, Fungal; D012342:RNA, Small Nuclear; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; D017411:Ribonucleoproteins, Small Nuclear; D029701:Saccharomyces cerevisiae Proteins