Targeted Identification of Protein Interactions in Eukaryotic mRNA Translation

Andrew J. Link*, Xinnan Niu, Connie M. Weaver, Jennifer L. Jennings, Dexter T. Duncan, K. Jill McAfee, Morgan Sammons, Vince R. Gerbasi, Adam R. Farley, Tracey C. Fleischer, Christopher M. Browne, Parimal Samir, Allison Galassie, Braden Boone

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract

To identify protein–protein interactions and phosphorylated amino acid sites in eukaryotic mRNA translation, replicate TAP-MudPIT and control experiments are performed targeting Saccharomyces cerevisiae genes previously implicated in eukaryotic mRNA translation by their genetic and/or functional roles in translation initiation, elongation, termination, or interactions with ribosomal complexes. Replicate tandem affinity purifications of each targeted yeast TAP-tagged mRNA translation protein coupled with multidimensional liquid chromatography and tandem mass spectrometry analysis are used to identify and quantify copurifying proteins. To improve sensitivity and minimize spurious, nonspecific interactions, a novel cross-validation approach is employed to identify the most statistically significant protein–protein interactions. Using experimental and computational strategies discussed herein, the previously described protein composition of the canonical eukaryotic mRNA translation initiation, elongation, and termination complexes is calculated. In addition, statistically significant unpublished protein interactions and phosphorylation sites for S. cerevisiae’s mRNA translation proteins and complexes are identified.

Original languageEnglish (US)
Article number1900177
JournalProteomics
Volume20
Issue number7
DOIs
StatePublished - Apr 1 2020
Externally publishedYes

Keywords

  • mRNA translation
  • phosphorylation
  • protein–protein interactions

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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