Abstract
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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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5' Untranslated Regions*
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Amino Acid Sequence
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DNA-Binding Proteins / physiology
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HeLa Cells
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Heterogeneous-Nuclear Ribonucleoproteins / antagonists & inhibitors
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Heterogeneous-Nuclear Ribonucleoproteins / chemistry
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Heterogeneous-Nuclear Ribonucleoproteins / physiology
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Humans
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Molecular Sequence Data
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Oligodeoxyribonucleotides / chemistry
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Oligodeoxyribonucleotides / metabolism
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Oligodeoxyribonucleotides / pharmacology*
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Poliovirus / genetics
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Protein Biosynthesis* / drug effects
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RNA-Binding Proteins / analysis*
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RNA-Binding Proteins / antagonists & inhibitors
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RNA-Binding Proteins / metabolism
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Rhinovirus / genetics
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Ribosomes / chemistry
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Ribosomes / genetics
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Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
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Transcription Factors / physiology
Substances
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5' Untranslated Regions
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DNA-Binding Proteins
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Heterogeneous-Nuclear Ribonucleoproteins
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Oligodeoxyribonucleotides
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PCBP1 protein, human
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RNA-Binding Proteins
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Transcription Factors