Proteomic analysis of reaper 5' untranslated region-interacting factors isolated by tobramycin affinity-selection reveals a role for La antigen in reaper mRNA translation.
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.
Vazquez-Pianzola
Paula
P
Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
Urlaub
Henning
H
Rivera-Pomar
Rolando
R
D020121:5' Untranslated Regions; D000818:Animals; D001324:Autoantigens / Q000378:metabolism*; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000235:genetics / Q000378:metabolism*; D004625:Embryo, Nonmammalian / Q000378:metabolism; D017930:Genes, Reporter; D013058:Mass Spectrometry; D014176:Protein Biosynthesis; D040901:Proteomics; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012261:Ribonucleoproteins / Q000378:metabolism*; D014020:Tissue Extracts / Q000378:metabolism; D014031:Tobramycin / Q000378:metabolism; D000097585:SS-B Antigen
D020121:5' Untranslated Regions; D001324:Autoantigens; D029721:Drosophila Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D012261:Ribonucleoproteins; D014020:Tissue Extracts; D014031:Tobramycin; D000097585:SS-B Antigen; C086973:rpr protein, Drosophila