Features of double-stranded RNA-binding domains of RNA helicase A are necessary for selective recognition and translation of complex mRNAs

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.

FIGURE 1.

RHA domain structure and isolation of recombinant N-term, C-term, and DEIH polypeptides. A, depiction of the 1269-amino acid RHA and amino acid coordinates of recombinant domains evaluated in this study; B, SDS-PAGE analysis of recombinant protein preparations.

FIGURE 2.

Conserved lysines in dsRBD1 and dsRBD2 are essential for effective binding of N-term to PCE RNA. Shown are the results from EMSAs of the indicated recombinant protein and ³²P-labeled RNA. A, wild-type N-term and ³²P-labeled junD PCE; B, wild-type N-term and ³²P-labeled SNV PCE; C, wild-type N-term and ³²P-labeled tRNA^Lys; D, K54A/K55A/K236E N-term and ³²P-labeled junD PCE; E, K54A/K55A/K236E N-term and ³²P-labeled SNV PCE; F, K54A/K55A N-term and ³²P-labeled SNV PCE; G, K236E N-term and ³²P-labeled SNV PCE.

FIGURE 3.

N-term exhibits specific interaction with PCE RNA of viral and cellular origin. Shown are the results from EMSA of N-term and ³²P-labeled SNV PCE RNA (0.02 μm) (lanes 1, 2, and 9–10) upon the addition of unlabeled SNV PCE (lanes 3–5 and 11–13), junD PCE (lanes 6–8), or tRNA^Lys (lanes 14–16). The concentrations of competitor RNA were 0.2, 1, and 2 μm, respectively. The protein concentration was 1 μm and shifted all ³²P-labeled SNV PCE RNA.

FIGURE 4.

N-term binding to PCE is recapitulated and quantified by FA measurements. Shown are the results from representative FA assays to measure the binding affinity of N-term for the indicated RNA. A, PCE^AB; B, PCE^AC; C, mutAC; D, minihelix^Lys; E, 7SL RNA. Anisotropy changes >0.04 are statistically significant.

FIGURE 5.

C-term lacks specificity for PCE RNA. Shown are the results from representative FA assays to measure the binding affinity of C-term for the indicated RNA. A, PCE^AB; B, PCE^AC; C, mutAC; D, minihelix^Lys; E, 7SL RNA.

FIGURE 6.

Mutation of conserved lysine residues eliminates affinity of N-term for PCE RNA. Shown are the results from representative FA assays to measure the binding affinity of the indicated N-term mutant protein and RNA. A, K54A/K55A/K236E N-term and PCE^AB; B, K54A/K55A/K236E N-term and PCE^AC; C, K54A/K55A N-term and PCE^AB; D, K54A/K55A N-term and PCE^AC; E, K236E N-term and PCE^AB; F, K236E N-term and PCE^AC.

FIGURE 7.

N-term interacts with PCE RNA in cells. Shown are the results from a representative immunoprecipitation (IP) assay to compare PCE RNA coprecipitation with N-term proteins. Empty FLAG plasmid, FLAG-tagged N-term, or FLAG-tagged K54A/K55A/K236E N-term was expressed in transfected HEK293 cells, and FLAG-tagged proteins were subjected to immunoprecipitation with anti-FLAG antibody (mouse) conjugated to agarose beads. Immunoblotting of equivalent aliquots of input lysate or immunoprecipitate on agarose beads determined that the immunoprecipitation efficiency was similar for each N-term protein. RNA was harvested from the remaining beads and subjected to RT-PCR with junD or GAPDH primer. A, Western blotting with FLAG-specific antiserum (rabbit) detected equivalent expression of N-term and K54A/K55A/K236E N-term. The 9-amino acid FLAG peptide was not resolved. B, Western blotting with FLAG-specific antiserum (rabbit) detected that immunoprecipitation was similar for N-term and K54A/K55A/K236E N-term. C, PCR amplification products from the indicated RNA preparation incubated with or without reverse transcriptase (RT+ or RT−, respectively). Representative samples of total RNA and RNA isolated from immunoprecipitated samples were evaluated with junD- or GAPDH-specific primers. The junD amplification product is 232 bp, and the lower band is the primer dimer.

FIGURE 8.

Exogenously expressed N-term dominantly inhibits translation of PCE reporter RNA. Shown are the results from a representative transfection assay to determine the translation activity of endogenous RHA upon exogenous expression of N-term. HEK293 cells were transfected with pYW100 SNV PCE reporter plasmid and increasing amounts of the indicated N-term expression plasmid. Total cell protein was subjected to Gag ELISA and Western blotting with anti-FLAG and anti-tubulin antisera. Total cellular RNA was subjected to real-time RT-PCR, and control reactions lacked reverse transcriptase. PCE reporter activity was determined in three independent assays summarized graphically as Gag protein relative to gag RNA standardized to actin. Error bars indicate S.D. *, statistically significant difference (p = < 0.03). A, PCE activity is inhibited by FLAG-tagged N-term. B, PCE activity is not changed by FLAG-tagged K54A/K55A/K236E N-term.