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Proteomic analysis of reaper 5' untranslated region‐interacting factors isolated by tobramycin affinity‐selection reveals a role for La antigen in reaper mRNA translation
Author(s) -
VazquezPianzola Paula,
Urlaub Henning,
RiveraPomar Rolando
Publication year - 2005
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200401045
Subject(s) - translation (biology) , messenger rna , biology , three prime untranslated region , untranslated region , selection (genetic algorithm) , computational biology , antigen , microbiology and biotechnology , genetics , gene , computer science , artificial intelligence
Translational control is a key step in gene expression regulation during apoptosis. To understand the mechanisms of mRNA translation of a pro‐apoptotic gene, reaper ( rpr ), we adapted the tobramycin‐aptamer technique described by Hartmuth et al. ( Proc. Natl. Acad. Sci. USA 2002, 99 , 16719–16724) for the analysis of proteins interacting with rpr 5' untranslated region (UTR). We assembled ribonucleoprotein complexes in vitro using translation extracts derived from Drosophila embryos and purified the RNA‐protein complexes for mas spectrometry analysis. We identified the proteins bound to the 5' UTR of rpr . One of them, the La antigen, was validated by RNA‐crosslinking experiments using recombinant protein and by the translation efficiency of reporter mRNAs in Drosophila cells after RNAinterference experiments. Our data provide evidence of the involvement of La antigen in the translation of rpr and set a protocol for purification of tagged‐RNA‐protein complexes from cytoplasmic extracts.