SLU7 and a novel activity, SSF1, act during the PRP16-dependent step of yeast pre-mRNA splicing.
Understanding the mechanism of pre-mRNA splicing requires the characterization of all components involved. In the present study, we used the genetically and biochemically defined yeast PRP16 protein as a point of departure for the identification of additional factors required for the second catalytic step in vitro. We isolated by glycerol gradient sedimentation spliceosomes that were formed in yeast extracts depleted of PRP16. This procedure separated the spliceosomal complexes containing lariat intermediate and exon 1 from free proteins present in the whole-cell yeast extract. We then supplemented these spliceosomes with purified proteins or yeast extract fractions as a functional assay for second-step splicing factors. We show that SLU7 protein and a novel activity that we named SSF1 (second-step factor 1) were required in concert with PRP16 to promote progression through the second catalytic step of splicing. Taking advantage of a differential ATP requirement for PRP16 and SLU7 function, we show that SLU7 can act after PRP16 in the splicing pathway.
Ansari
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Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA.
Schwer
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D000199:Actins / Q000235:genetics; D000251:Adenosine Triphosphatases / Q000302:isolation & purification / Q000502:physiology*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D002458:Cell Fractionation; D005656:Fungal Proteins / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D005800:Genes, Fungal / Q000235:genetics; D008969:Molecular Sequence Data; D020365:RNA Helicases; D012322:RNA Precursors / Q000235:genetics; D012326:RNA Splicing / Q000502:physiology*; D000072260:RNA Splicing Factors; D012331:RNA, Fungal / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics*; D016601:RNA-Binding Proteins / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000502:physiology; D017411:Ribonucleoproteins, Small Nuclear; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D017381:Spliceosomes / Q000378:metabolism
D000199:Actins; D005656:Fungal Proteins; D012322:RNA Precursors; D000072260:RNA Splicing Factors; D012331:RNA, Fungal; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D017411:Ribonucleoproteins, Small Nuclear; C095479:SLU7 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C075669:PRP16 protein, S cerevisiae; D020365:RNA Helicases