Identification of cellular proteins enhancing activities of internal ribosomal entry sites by competition with oligodeoxynucleotides.
The translation of numerous eukaryotic mRNAs is mediated by internal ribosomal entry sites (IRESs). IRES-dependent translation requires both canonical translation initiation factors and IRES-specific trans-acting factors (ITAFs). Here we report a strategy to identify and characterize ITAFs required for IRES-dependent translation. This process involves steps for identifying oligodeoxynucleotides affecting IRES-dependent translation, purifying proteins interacting with the inhibitory DNA, identifying the specific proteins with matrix-assisted laser desorption ionization/time-of-flight mass spectrometry, and confirming the roles of these proteins in IRES-dependent translation by depletion and repletion of proteins from an in vitro translation system. Using this strategy, we show that poly(rC)-binding proteins 1 and 2 enhance translation through polioviral and rhinoviral IRES elements.
Choi
Kobong
K
Department of Life Science, Division of Molecular and Life Sciences, Pohang University of Science and Technology, San 31, Hyoja-Dong, Pohang, Kyungbuk 790-784, Korea.
Kim
Jong Heon
JH
Li
Xiaoyu
X
Paek
Ki Young
KY
Ha
Sang Hoon
SH
Ryu
Sung Ho
SH
Wimmer
Eckard
E
Jang
Sung Key
SK
D020121:5' Untranslated Regions; D000595:Amino Acid Sequence; D004268:DNA-Binding Proteins / Q000502:physiology; D006367:HeLa Cells; D034441:Heterogeneous-Nuclear Ribonucleoproteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000502:physiology; D006801:Humans; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D017955:Poliovirus / Q000235:genetics; D014176:Protein Biosynthesis / Q000187:drug effects; D016601:RNA-Binding Proteins / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism; D012229:Rhinovirus / Q000235:genetics; D012270:Ribosomes / Q000737:chemistry / Q000235:genetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014157:Transcription Factors / Q000502:physiology
D020121:5' Untranslated Regions; D004268:DNA-Binding Proteins; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D009838:Oligodeoxyribonucleotides; C094604:PCBP1 protein, human; D016601:RNA-Binding Proteins; D014157:Transcription Factors