Yeast ortholog of the Drosophila crooked neck protein promotes spliceosome assembly through stable U4/U6.U5 snRNP addition.
Mutants in the Drosophila crooked neck (crn) gene show an embryonic lethal phenotype with severe developmental defects. The unusual crn protein consists of sixteen tandem repeats of the 34 amino acid tetratricopeptide (TPR) protein recognition domain. Crn-like TPR elements are found in several RNA processing proteins, although it is unknown how the TPR repeats or the crn protein contribute to Drosophila development. We have isolated a Saccharomyces cerevisiae gene, CLF1, that encodes a crooked neck-like factor. CLF1 is an essential gene but the lethal phenotype of a clf1::HIS3 chromosomal null mutant can be rescued by plasmid-based expression of CLF1 or the Drosophila crn open reading frame. Clf1p is required in vivo and in vitro for pre-mRNA 5' splice site cleavage. Extracts depleted of Clf1p arrest spliceosome assembly after U2 snRNP addition but prior to productive U4/U6.U5 association. Yeast two-hybrid analyses and in vitro binding studies show that Clf1p interacts specifically and differentially with the U1 snRNP-Prp40p protein and the yeast U2AF65 homolog, Mud2p. Intriguingly, Prp40p and Mud2p also bind the phylogenetically conserved branchpoint binding protein (BBP/SF1). Our results indicate that Clf1p acts as a scaffolding protein in spliceosome assembly and suggest that Clf1p may support the cross-intron bridge during the prespliceosome-to-spliceosome transition.
Chung
S
S
T.H. Morgan School of Biological Sciences and the Markey Cancer Center, University of Kentucky, Lexington 40506-0225, USA.
McLean
M R
MR
Rymond
B C
BC
D000595:Amino Acid Sequence; D000818:Animals; D018797:Cell Cycle Proteins; D002846:Chromatography, Affinity; D004330:Drosophila / Q000235:genetics*; D029721:Drosophila Proteins; D005656:Fungal Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D019476:Insect Proteins / Q000235:genetics*; D008957:Models, Genetic; D008969:Molecular Sequence Data; D010641:Phenotype; D012326:RNA Splicing; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000378:metabolism; D017414:Ribonucleoprotein, U4-U6 Small Nuclear / Q000494:pharmacology*; D017415:Ribonucleoprotein, U5 Small Nuclear / Q000494:pharmacology*; D012261:Ribonucleoproteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D017381:Spliceosomes / Q000378:metabolism*; D000072668:Splicing Factor U2AF; D013997:Time Factors
C419763:CLF1 protein, S cerevisiae; D018797:Cell Cycle Proteins; D029721:Drosophila Proteins; D005656:Fungal Proteins; D019476:Insect Proteins; C089980:MUD2 protein, S cerevisiae; C120813:Prp40 protein, S cerevisiae; D017412:Ribonucleoprotein, U1 Small Nuclear; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; D017415:Ribonucleoprotein, U5 Small Nuclear; D012261:Ribonucleoproteins; D029701:Saccharomyces cerevisiae Proteins; D000072668:Splicing Factor U2AF; C069204:crn protein, Drosophila