Journal
RNA
Volume 24, Issue 7, Pages 982-989Publisher
COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT
DOI: 10.1261/rna.065730.118
Keywords
in vitro translation; RNA affinity purification; mRNP; Pseudomonas phage 7; RNA-binding protein; RNP purification
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Funding
- Intramural Research Program, National Institutes of Health, National Heart, Lung, and Blood Institute
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Cell-free systems are widely used to study mechanisms and regulation of translation, but the use of in vitro transcribed (IVT) mRNAs as translation substrates limits their efficiency and utility. Here, we present an approach for in vitro translation of messenger ribonucleoprotein (mRNP) complexes affinity purified in association with tagged mRNAs expressed in mammalian cells. We show that in vitro translation of purified mRNPs is much more efficient than that achieved using standard IVT mRNA substrates and is compatible with physiological ionic conditions. The high efficiency of affinity-purified mRNP in vitro translation is attributable to both copurified protein components and proper mRNA processing and modification. Further, we use translation inhibitors to show that translation of purified mRNPs consists of separable phases of run-off elongation by copurified ribosomes and de novo initiation by ribosomes present in the translation extracts. We expect that this in vitro system will enhance mechanistic studies of eukaryotic translation and translation-associated processes by allowing the use of endogenous mRNPs as translation substrates under physiological buffer conditions.
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