A systematic characterization of Cwc21, the yeast ortholog of the human spliceosomal protein SRm300.
Cwc21 (complexed with Cef1 protein 21) is a 135 amino acid yeast protein that shares homology with the N-terminal domain of human SRm300/SRRM2, a large serine/arginine-repeat protein shown previously to associate with the splicing coactivator and 3'-end processing stimulatory factor, SRm160. Proteomic analysis of spliceosomal complexes has suggested a role for Cwc21 and SRm300 at the core of the spliceosome. However, specific functions for these proteins have remained elusive. In this report, we employ quantitative genetic interaction mapping, mass spectrometry of tandem affinity-purified complexes, and microarray profiling to investigate genetic, physical, and functional interactions involving Cwc21. Combined data from these assays support multiple roles for Cwc21 in the formation and function of splicing complexes. Consistent with a role for Cwc21 at the core of the spliceosome, we observe strong genetic, physical, and functional interactions with Isy1, a protein previously implicated in the first catalytic step of splicing and splicing fidelity. Together, the results suggest multiple functions for Cwc21/SRm300 in the splicing process, including an important role in the activation of splicing in association with Isy1.
Banting and Best Department of Medical Research, Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario M5S 3E1, Canada.
May
M
Khanna
Harm
H
Van Bakel
Xinyi
X
Tang
John A
JA
Calarco
Tomas
T
Babak
Grace
G
Guo
Andrew
A
Emili
Jack F
JF
Greenblatt
Timothy R
TR
Hughes
Nevan J
NJ
Krogan
Benjamin J
BJ
Blencowe
D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis; D011485:Protein Binding; D040901:Proteomics; D012326:RNA Splicing; D012342:RNA, Small Nuclear / Q000235:genetics; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D017381:Spliceosomes / Q000378:metabolism*
D002352:Carrier Proteins; C545658:Cwc21 protein, S cerevisiae; C118319:ISY1 protein, S cerevisiae; D012342:RNA, Small Nuclear; D016601:RNA-Binding Proteins; C414618:SRRM2 protein, human; D029701:Saccharomyces cerevisiae Proteins