Testing the faux-UTR model for NMD: analysis of Upf1p and Pab1p competition for binding to eRF3/Sup35p.
Nonsense-mediated mRNA decay (NMD) is a surveillance mechanism that accelerates the degradation of mRNAs containing premature translation termination codons. This quality control pathway depends on the NMD-specific factors, Upf1p, Upf2p/Nmd2p, and Upf3p, as well as the two release factors, eRF1 and eRF3 (respectively designated Sup45p and Sup35p in yeast). NMD activation is also enabled by the absence of the poly(A)-binding protein, Pab1p, downstream of a termination event. Since Sup35p interacts with both Upf1p and Pab1p we considered the possibility that differential binding of the latter factors to Sup35p may be a critical determinant of NMD sensitivity for an mRNA. Here we describe three approaches to assess this hypothesis. First, we tethered fragments or mutant forms of Sup35p downstream of a premature termination codon in a mini-pgk1 nonsense-containing mRNA and showed that the inhibition of NMD by tethered Sup35p does not depend on the domain necessary for the recruitment of Pab1p. Second, we examined the Sup35p interaction properties of Upf1p and Pab1p in vitro and showed that these two proteins bind differentially to Sup35p. Finally, we examined competitive binding between the three proteins and observed that Upf1p inhibits Pab1p binding to Sup35p whereas the interaction between Upf1p and Sup35p is relatively unaffected by Pab1p. These data indicate that the binding of Upf1p and Pab1p to Sup35p may be more complex than anticipated and that NMD activation could involve more than just simple competition between these factors. We conclude that activation of NMD at a premature termination codon is not solely based on the absence of Pab1p and suggest that a specific recruitment step must commit Upf1p to the process and Upf1p-associated mRNAs to NMD.
CNRS UPR9073 Associated with Université Paris Diderot, Sorbonne Paris Cité, Institut de Biologie Physico-chimique (IBPC) 13 rue Pierre et Marie Curie, 75005 Paris, France.
Stephanie
S
Kervestin
Chunfang
C
Li
Richard
R
Buckingham
Allan
A
Jacobson
D020413:3' Untranslated Regions; D001667:Binding, Competitive; D018388:Codon, Terminator; D017930:Genes, Reporter / Q000235:genetics; D008954:Models, Biological; D009154:Mutation; D059365:Nonsense Mediated mRNA Decay; D010454:Peptide Termination Factors / Q000737:chemistry / Q000378:metabolism; D039102:Poly(A)-Binding Proteins / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D020365:RNA Helicases / Q000378:metabolism; D020871:RNA Stability; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*
D020413:3' Untranslated Regions; D018388:Codon, Terminator; D010454:Peptide Termination Factors; D039102:Poly(A)-Binding Proteins; D012333:RNA, Messenger; C068068:SUP35 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C450100:pab1 protein, S cerevisiae; C087971:peptide-chain-release factor 3; C074151:NAM7 protein, S cerevisiae; D020365:RNA Helicases