Cotranscriptional spliceosome assembly and splicing are independent of the Prp40p WW domain.
Complex cellular functions involve large networks of interactions. Pre-mRNA splicing and transcription are thought to be coupled by the C-terminal domain (CTD) of the large subunit of RNA polymerase II (Pol II). In yeast, the U1 snRNP subunit Prp40 was proposed to mediate cotranscriptional recruitment of early splicing factors through binding of its WW domains to the Pol II CTD. Here we investigate the role of Prp40 in splicing with an emphasis on the role of the WW domains, which might confer protein-protein interactions among the splicing and transcriptional machineries. Affinity purification revealed that Prp40 and Snu71 form a stable heterodimer that stably associates with the U1 snRNP only in the presence of Nam8, a known regulator of 5' splice site recognition. However, the Prp40 WW domains were dispensable for yeast viability. In their absence, no defect in splicing in vivo, U1 or U2 snRNP recruitment in vivo, or early splicing complex assembly in vitro was detected. We conclude that the WW domains of Prp40 do not mediate essential coupling between U1 snRNP and Pol II. Instead, delays in cotranscriptional U5 snRNP and Prp19 recruitment and altered spliceosome formation in vitro suggest that Prp40 WW domains assist in late steps of spliceosome assembly.
Max Planck Institute of Molecular Cell Biology and Genetics, 01307 Dresden, Germany.
Janina
J
Görnemann
Charlotte
C
Barrandon
Katja
K
Hujer
Berthold
B
Rutz
Guillaume
G
Rigaut
Kimberly M
KM
Kotovic
Céline
C
Faux
Karla M
KM
Neugebauer
Bertrand
B
Séraphin
D015966:Gene Expression Regulation, Fungal; D006801:Humans; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism; D012326:RNA Splicing; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017381:Spliceosomes / Q000378:metabolism*; D014158:Transcription, Genetic; D015003:Yeasts / Q000235:genetics / Q000378:metabolism
D012322:RNA Precursors; D017412:Ribonucleoprotein, U1 Small Nuclear