A targeted bypass screen identifies Ynl187p, Prp42p, Snu71p, and Cbp80p for stable U1 snRNP/Pre-mRNA interaction.
To understand how DEXD/H-box proteins recognize and interact with their cellular substrates, we have been studying Prp28p, a DEXD/H-box splicing factor required for switching the U1 snRNP with the U6 snRNP at the precursor mRNA (pre-mRNA) 5' splice site. We previously demonstrated that the requirement for Prp28p can be eliminated by mutations that alter either the U1 snRNA or the U1C protein, suggesting that both are targets of Prp28p. Inspired by this finding, we designed a bypass genetic screen to specifically search for additional, novel targets of Prp28p. The screen identified Prp42p, Snu71p, and Cbp80p, all known components of commitment complexes, as well as Ynl187p, a protein of uncertain function. To examine the role of Ynl187p in splicing, we carried out extensive genetic and biochemical analysis, including chromatin immunoprecipitation. Our data suggest that Ynl187p acts in concert with U1C and Cbp80p to help stabilize the U1 snRNP-5' splice site interaction. These findings are discussed in the context of DEXD/H-box proteins and their role in vivo as well as the potential need for more integral U1-snRNP proteins in governing the fungal 5' splice site RNA-RNA interaction compared to the number of U1 snRNP proteins needed by metazoans.
Hage
Rosemary
R
Department of Molecular Genetics, The Ohio State University, 484 West 12th Ave., Columbus, OH 43210, USA.
Tung
Luh
L
Du
Hansen
H
Stands
Leah
L
Rosbash
Michael
M
Chang
Tien-Hsien
TH
D000595:Amino Acid Sequence; D001483:Base Sequence; D053487:DEAD-box RNA Helicases / Q000235:genetics / Q000378:metabolism; D004271:DNA, Fungal / Q000235:genetics; D005800:Genes, Fungal; D016153:Genes, Suppressor; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D039381:RNA Cap-Binding Proteins; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism*; D022821:RNA Splice Sites; D012331:RNA, Fungal / Q000235:genetics / Q000378:metabolism*; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
D004271:DNA, Fungal; D009687:Nuclear Proteins; D039381:RNA Cap-Binding Proteins; D012322:RNA Precursors; D022821:RNA Splice Sites; D012331:RNA, Fungal; D017412:Ribonucleoprotein, U1 Small Nuclear; C101398:STO1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C088939:PRP28 protein, S cerevisiae; D053487:DEAD-box RNA Helicases