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PMID: 10445879 Published · ppublish English Journal Article Research Support, U.S. Gov't, P.H.S.

Yeast ortholog of the Drosophila crooked neck protein promotes spliceosome assembly through stable U4/U6.U5 snRNP addition.

RNA (New York, N.Y.) ·Vol. 5 ·No. 8 ·1999-08-00 ·Pages 1042-54

Chung S, McLean MR, Rymond BC

Abstract

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.

MeSH Terms
Amino Acid Sequence Animals Cell Cycle Proteins Chromatography, Affinity Drosophila/genetics Drosophila Proteins Fungal Proteins/genetics,isolation & purification,metabolism Insect Proteins/genetics Models, Genetic Molecular Sequence Data Phenotype RNA Splicing Ribonucleoprotein, U1 Small Nuclear/metabolism Ribonucleoprotein, U4-U6 Small Nuclear/pharmacology Ribonucleoprotein, U5 Small Nuclear/pharmacology Ribonucleoproteins/metabolism Saccharomyces cerevisiae/genetics Saccharomyces cerevisiae Proteins Sequence Homology, Amino Acid Spliceosomes/metabolism Splicing Factor U2AF Time Factors
Chemicals
CLF1 protein, S cerevisiae Cell Cycle Proteins Drosophila Proteins Fungal Proteins Insect Proteins MUD2 protein, S cerevisiae Prp40 protein, S cerevisiae Ribonucleoprotein, U1 Small Nuclear Ribonucleoprotein, U4-U6 Small Nuclear Ribonucleoprotein, U5 Small Nuclear Ribonucleoproteins Saccharomyces cerevisiae Proteins Splicing Factor U2AF crn protein, Drosophila
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Chung S
T.H. Morgan School of Biological Sciences and the Markey Cancer Center, University of Kentucky, Lexington 40506-0225, USA.
McLean M R
Rymond B C
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Article Info
Journal
RNA (New York, N.Y.)
Abbr.
RNA
ISSN
1355-8382
DOI
10.1017/s1355838299990635
Published
1999-08-00
Pages
1042-54
Language
English
Region
United States
NLM ID
9509184
PMCID
PMC1369828
Subset
IM
Grants
NIGMS NIH HHS · GM42476 · United States
External Links
PubMed source DOI: 10.1017/s1355838299990635 PMC Full Text View journal details
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