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Highly efficient in vitro translation of authentic affinity-purified messenger ribonucleoprotein complexes.

ABSTRACT: 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.

SUBMITTER: Fritz SE

PROVIDER: S-EPMC6004058 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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Highly efficient in vitro translation of authentic affinity-purified messenger ribonucleoprotein complexes.

Fritz Sarah E SE Haque Nazmul N Hogg J Robert JR

RNA (New York, N.Y.) 20180503 7

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 sub ...[more]

PMID: 29724884

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Project description:The 3'-UTR (untranslated region) of bcl-2 mRNA contains an ARE (AU-rich element) that potentially regulates the stability of bcl-2 mRNA in a cell specific fashion. Previous studies have demonstrated that multiple proteins interact with bcl-2 mRNA in HL-60 (human leukaemia-60) cells, potentially contributing to the overexpression of Bcl-2 protein. Treatment of HL-60 cells with taxol or okadaic acid has been shown to induce destabilization of bcl-2 mRNA, which was associated with decreased binding of trans-acting factors to bcl-2 mRNA. Nucleolin has been identified as one of the bcl-2 mRNA-binding proteins [Sengupta, Bandyopadhyay, Fernandes and Spicer (2004) J. Biol. Chem. 279, 10855-10863]. In an effort to identify additional bcl-2 mRNA-binding proteins, two polypeptides of approx. 45 kDa and 60 kDa were isolated from HL-60 cells by ARE(bcl-2) (transcripts that contain bcl-2 AREs) RNA affinity chromatography. These proteins were identified as the human proliferation associated protein, Ebp1, and human DRBP76 (double stranded RNA-binding protein 76) respectively, by MALDI (matrix-assisted laser-desorption ionization)-MS. RNA electrophoretic mobility shift assays indicated that recombinant Ebp1 binds to ARE(bcl-2) RNA but not to the group 1 ARE present in GM-CSF (granulocyte macrophage-colony stimulating factor) mRNA in vitro. Antibody supershift assays demonstrated that Ebp1 is present in protein-ARE(bcl-2) RNA complexes formed with cytosolic HL-60 extracts. The interaction of Ebp1 with bcl-2 mRNA in HL-60 cells was also demonstrated by RNA co-immunoprecipitation assays. This interaction was not detected in extracts of taxol-treated HL-60 cells. Immunoprecipitation assays further revealed that Ebp1 co-precipitates with nucleolin from HL-60 cytoplasmic extracts. The observation that co-precipitation was decreased when extracts were treated with RNase suggests that Ebp1 and nucleolin are present in the same bcl-2 mRNP (messenger ribonucleoprotein particle) complexes. RNA-decay assays further demonstrated that Ebp1 decreased the rate of decay of beta-globin-ARE(bcl-2) transcripts in HL-60 cell extracts. Collectively, these results indicate a novel function for Ebp1 in contributing to the regulation of bcl-2 expression in HL-60 cells.

Dataset Information

Highly efficient in vitro translation of authentic affinity-purified messenger ribonucleoprotein complexes.

Publications

Highly efficient in vitro translation of authentic affinity-purified messenger ribonucleoprotein complexes.

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