title: Composition of yeast snRNPs and snoRNPs in the absence of trimethylguanosine caps reveals nuclear cap binding protein as a gained U1 component implicated in the cold-sensitivity of tgs1Δ cells.
abstract: Small nuclear and nucleolar RNAs that program pre-mRNA splicing and rRNA processing have a signature 5'-trimethylguanosine (TMG) cap. Whereas the mechanism of TMG synthesis by Tgs1 methyltransferase has been elucidated, we know little about whether or how RNP biogenesis, structure and function are perturbed when TMG caps are missing. Here, we analyzed RNPs isolated by tandem-affinity purification from TGS1 and tgs1Δ yeast strains. The protein and U-RNA contents of total SmB-containing RNPs were similar. Finer analysis revealed stoichiometric association of the nuclear cap-binding protein (CBP) subunits Sto1 and Cbc2 with otherwise intact Mud1- and Nam8-containing U1 snRNPs from tgs1Δ cells. CBP was not comparably enriched in Lea1-containing U2 snRNPs from tgs1Δ cells. Moreover, CBP was not associated with mature Nop58-containing C/D snoRNPs or mature Cbf5- and Gar1-containing H/ACA snoRNPs from tgs1Δ cells. The protein composition and association of C/D snoRNPs with the small subunit (SSU) processosome were not grossly affected by absence of TMG caps, nor was the composition of H/ACA snoRNPs. The cold-sensitive (cs) growth defect of tgs1Δ yeast cells could be suppressed by mutating the cap-binding pocket of Cbc2, suggesting that ectopic CBP binding to the exposed U1 m(7)G cap in tgs1Δ cells (not lack of TMG caps per se) underlies the cs phenotype.
authors.affiliation: Department of Microbiology and Immunology, Weill Cornell Medical College, New York, NY 10065, USA. bschwer@med.cornell.edu
authors.forename: Beate
authors.initials: B
authors.lastname: Schwer
authors.forename: Hediye
authors.initials: H
authors.lastname: Erdjument-Bromage
authors.forename: Stewart
authors.initials: S
authors.lastname: Shuman
mesh_terms: D001324:Autoantigens / Q000302:isolation & purification; D003080:Cold Temperature; D017353:Gene Deletion; D008780:Methyltransferases / Q000235:genetics*; D039502:Nuclear Cap-Binding Protein Complex / Q000032:analysis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D010641:Phenotype; D012315:RNA Caps / Q000378:metabolism; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000737:chemistry / Q000302:isolation & purification; D017413:Ribonucleoprotein, U2 Small Nuclear / Q000737:chemistry / Q000302:isolation & purification; D017411:Ribonucleoproteins, Small Nuclear / Q000737:chemistry* / Q000302:isolation & purification; D020718:Ribonucleoproteins, Small Nucleolar / Q000737:chemistry* / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013489:Suppression, Genetic
chemical_list: D001324:Autoantigens; C100383:CBC2 protein, S cerevisiae; C116361:LEA1 protein, S cerevisiae; D039502:Nuclear Cap-Binding Protein Complex; D012315:RNA Caps; D017412:Ribonucleoprotein, U1 Small Nuclear; D017413:Ribonucleoprotein, U2 Small Nuclear; D017411:Ribonucleoproteins, Small Nuclear; D020718:Ribonucleoproteins, Small Nucleolar; C489392:SMB protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D008780:Methyltransferases; C456271:trimethylguanosine synthase