Abstract
The DExH protein RNA helicase A (RHA) plays numerous roles in cell physiology, and post-transcriptional activation of gene expression is a major role among them. RHA selectively activates translation of complex cellular and retroviral mRNAs. Although RHA requires interaction with structural features of the 5'-UTR of these target mRNAs, the molecular basis of their translation activation by RHA is poorly understood. RHA contains a conserved ATPase-dependent helicase core that is flanked by two α-β-β-β-α double-stranded RNA-binding domains at the N terminus and repeated arginine-glycine residues at the C terminus. The individual recombinant N-terminal, central helicase, and C-terminal domains were evaluated for their ability to specifically interact with cognate RNAs by in vitro biochemical measurements and mRNA translation assays in cells. The results demonstrate that N-terminal residues confer selective interaction with retroviral and junD target RNAs. Conserved lysine residues in the distal α-helix of the double-stranded RNA-binding domains are necessary to engage structural features of retroviral and junD 5'-UTRs. Exogenous expression of the N terminus coprecipitates junD mRNA and inhibits the translation activity of endogenous RHA. The results indicate that the molecular basis for the activation of translation by RHA is recognition of target mRNA by the N-terminal domain that tethers the ATP-dependent helicase for rearrangement of the complex 5'-UTR.
Highlights
RNA helicase A (RHA)2 is a ubiquitous DEIH superfamily 2 helicase that is necessary for translation of retroviral and selected cellular mRNAs [1,2,3]
The results indicate that the molecular basis for the activation of translation by RHA is recognition of target mRNA by the N-terminal domain that tethers the ATP-dependent helicase for rearrangement of the complex 5-UTR
The helicase domain is necessary for RHA translation activity on post-transcriptional control element (PCE) target mRNA [3]
Summary
RNA Transcription—In vitro transcripts were generated in the RiboMAXTM large-scale RNA production system (Promega) in the presence of [␣-32P]UTP/[␣-32P]CTP (PerkinElmer Life Sciences), treated with DNase (Promega), separated on 8% denaturing urea gels, and eluted in passive gel elution buffer (Ambion). EMSA—EMSAs were performed as described [1] with recombinant protein and 100,000 cpm of in vitro transcribed ␣-32P-labeled RNA (see supplemental “Experimental Procedures”) in EMSA buffer (2% glycerol, 0.8 mM EGTA, 0.2 mM EDTA, 2 mM Tris, pH 7.6, 14 mM KCl, and 0.2 mM Mg(OAc)). Cells were harvested in PBS and resuspended in 100 l of polysome buffer (100 mM KCl, 5 mM MgCl2, 10 mM HEPES, pH 7.0, 0.5% Nonidet P-40, and 1 mM DTT) supplemented with RNaseOUT (Invitrogen) and a protease inhibitor mixture of serine, cysteine, and aspartic proteases and aminopeptidase (Sigma). The soluble lysate was incubated with anti-FLAG monoclonal antibody M2-conjugated agarose beads (Sigma) that were equilibrated in NT2 buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM MgCl2, and 0.05% Nonidet P-40) overnight at 4 °C. RNA was detected by real-time RT-PCR analysis with gene-specific primers (KB1614/KB1615 for gag and KB1252/KB1253 for actin) (supplemental Table S1) [3]
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