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.
Subject(s)
Methyltransferases/genetics , Nuclear Cap-Binding Protein Complex/analysis , Ribonucleoproteins, Small Nuclear/chemistry , Ribonucleoproteins, Small Nucleolar/chemistry , Saccharomyces cerevisiae/genetics , Autoantigens/isolation & purification , Cold Temperature , Gene Deletion , Nuclear Cap-Binding Protein Complex/chemistry , Nuclear Cap-Binding Protein Complex/genetics , Nuclear Cap-Binding Protein Complex/isolation & purification , Phenotype , RNA Caps/metabolism , Ribonucleoprotein, U1 Small Nuclear/chemistry , Ribonucleoprotein, U1 Small Nuclear/isolation & purification , Ribonucleoprotein, U2 Small Nuclear/chemistry , Ribonucleoprotein, U2 Small Nuclear/isolation & purification , Ribonucleoproteins, Small Nuclear/isolation & purification , Ribonucleoproteins, Small Nucleolar/isolation & purification , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/isolation & purification , Suppression, GeneticABSTRACT
Nonsense-mediated mRNA decay (NMD) is one of the mRNA surveillance mechanisms, which eliminates aberrant mRNAs harboring premature termination codons. NMD targets only mRNAs bound by the nuclear cap-binding protein complex CBP80/20 which directs the pioneer round of translation. Here we demonstrate that NMD occurs efficiently during prolonged hypoxia in which steady-state translation is drastically inhibited. Accordingly, CBP80 remains in the nucleus, and processing bodies are unaffected with regard to their abundance and number under prolonged hypoxic conditions. These results indicate that mRNAs enter the pioneer round of translation during prolonged hypoxia.
