Mass spectrometric identification of proteins that interact through specific domains of the poly(A) binding protein.

Roy Richardson, Clyde L Denis, Chongxu Zhang, Maria E O Nielsen, Yueh-Chin Chiang, Morten Kierkegaard, Xin Wang, Darren J Lee, Jens S Andersen, Gang Yao

Molecular genetics and genomics : MGG 2012 Sep

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Poly(A) binding protein (PAB1) is involved in a number of RNA metabolic functions in eukaryotic cells and correspondingly is suggested to associate with a number of proteins. We have used mass spectrometric analysis to identify 55 non-ribosomal proteins that specifically interact with PAB1 from Saccharomyces cerevisiae. Because many of these factors may associate only indirectly with PAB1 by being components of the PAB1-mRNP structure, we additionally conducted mass spectrometric analyses on seven metabolically defined PAB1 deletion derivatives to delimit the interactions between these proteins and PAB1. These latter analyses identified 13 proteins whose associations with PAB1 were reduced by deleting one or another of PAB1's defined domains. Included in this list of 13 proteins were the translation initiation factors eIF4G1 and eIF4G2, translation termination factor eRF3, and PBP2, all of whose previously known direct interactions with specific PAB1 domains were either confirmed, delimited, or extended. The remaining nine proteins that interacted through a specific PAB1 domain were CBF5, SLF1, UPF1, CBC1, SSD1, NOP77, yGR250c, NAB6, and GBP2. In further study, UPF1, involved in nonsense-mediated decay, was confirmed to interact with PAB1 through the RRM1 domain. We additionally established that while the RRM1 domain of PAB1 was required for UPF1-induced acceleration of deadenylation during nonsense-mediated decay, it was not required for the more critical step of acceleration of mRNA decapping. These results begin to identify the proteins most likely to interact with PAB1 and the domains of PAB1 through which these contacts are made.

Citation

Roy Richardson, Clyde L Denis, Chongxu Zhang, Maria E O Nielsen, Yueh-Chin Chiang, Morten Kierkegaard, Xin Wang, Darren J Lee, Jens S Andersen, Gang Yao. Mass spectrometric identification of proteins that interact through specific domains of the poly(A) binding protein. Molecular genetics and genomics : MGG. 2012 Sep;287(9):711-730