DENR-MCTS1 heterodimerization and tRNA recruitment are required for translation reinitiation

Abstract

The succession of molecular events leading to eukaryotic translation reinitiation-whereby ribosomes terminate translation of a short open reading frame (ORF), resume scanning, and then translate a second ORF on the same mRNA-is not well understood. Density-regulated reinitiation and release factor (DENR) and multiple copies in T-cell lymphoma-1 (MCTS1) are implicated in promoting translation reinitiation both in vitro in translation extracts and in vivo. We present here the crystal structure of MCTS1 bound to a fragment of DENR. Based on this structure, we identify and experimentally validate that DENR residues Glu42, Tyr43, and Tyr46 are important for MCTS1 binding and that MCTS1 residue Phe104 is important for tRNA binding. Mutation of these residues reveals that DENR-MCTS1 dimerization and tRNA binding are both necessary for DENR and MCTS1 to promote translation reinitiation in human cells. These findings thereby link individual residues of DENR and MCTS1 to specific molecular functions of the complex. Since DENR-MCTS1 can bind tRNA in the absence of the ribosome, this suggests the DENR-MCTS1 complex could recruit tRNA to the ribosome during reinitiation analogously to the eukaryotic initiation factor 2 (eIF2) complex in cap-dependent translation.

Fig 1. High-resolution structure of MCTS1 binding an N-terminal fragment of DENR.

(A) Domain architecture of DENR, MCTS1, and the related eIF2D (ligatin) protein. (B) A DENR truncation series identifies aas 24–51 of DENR as the minimum peptide capable of binding MCTS1 in Escherichia coli. Summary of data presented in S1 Fig. (C) Crystal structure of the minimal DENR–MCTS1 complex. MCTS1 contains an N-terminal DUF1947 (light green) and a C-terminal PUA domain (green). The DENR peptide (light orange) binds along the interface between the N- and C-terminal MCTS1 domains. Anomalous density (14σ), calculated with ANODE, is shown as a mesh. (D) DENR contains a zinc finger at the N-terminus, comprised of Cys34, 37, and 44. Although our DENR construct lacks a fourth residue (Cys53) to complete the Zn²⁺ coordination, the zinc finger is properly folded, as it is completed by His58 of a crystallographically related MCTS1 molecule. aa, amino acid; DENR, density-regulated reinitiation and release factor; DUF1947, domain of unknown function 1947; eIF1, eukaryotic initiation factor 1; eIF2D, eukaryotic initiation factor 2D; MCTS1, multiple copies in T-cell lymphoma-1; MDM2, mouse double minute 2; PUA, pseudouridine synthase and archaeosine transglycosylase; SUI1, suppressors of initiation codon mutations 1; SWIB SWItch/Sucrose Nonfermentable complex B; WH, winged helix

Fig 2. DENR–MCTS1 binding is required for function of the complex.

(A) Residues involved in the interaction between DENR and MCTS1. DENR-Glu42 interacts with the nitrogen of the main chain of MCTS1-Gln140 and His141. Furthermore, DENR-Tyr43 and Tyr46 hydrogen-bond with MCTS1-His141 and Lys139, respectively. (B) Mutation of DENR E42, Y43, and Y46—but not any two of these three—abolishes binding to MCTS1 in E. coli. HIS-tagged DENR variants were coexpressed in E. coli and purified by affinity chromatography to detect copurifying MCTS1 (representative of 4 biological replicates). (C) Triple DENR mutation E42R, Y43A, Y46A (“RAA”) abolishes binding of DENR to MCTS1 in HeLa cells. HeLa cells were transfected to express DENR[WT] (“WT”), DENR[RAA] (“RAA”), or neither (“-“). DENR–MCTS1 binding was assayed by immunoprecipitating HA-tagged DENR variants and detecting coprecipitating endogenous MCTS1 by immunoblot representative of 2 biological replicates). (D-D') DENR must bind MCTS1 to be functionally active. (D-D”) Activity of DENR[E42R, Y43A, Y46A] (“RAA”) assayed by reconstituting DENR-knockdown HeLa cells by reexpressing (“OE”) either WT or mutant DENR. DENR[WT] and DENR[RAA] contain synonymous substitutions to avoid siRNA-mediated knockdown. Activity is assayed as the ability to promote translation reinitiation downstream of a stuORF as previously reported [10] (n = 3). (D”) Since DENR[RAA] is less stable than DENR[WT] (see S2D Fig), the amount of DENR[RAA] overexpression plasmid was elevated to yield at least as much protein as DENR[WT]. Shown here are DENR levels from the same set of cells as in (D’). Underlying data available in S1 Data. CMV, cytomegalovirus; DENR, density-regulated reinitiation and release factor; FLUC, firefly luciferase; HA, human influenza hemagglutinin; HIS, polyhistidine; IP, immunoprecipitated; MCTS1, multiple copies in T-cell lymphoma-1; RLUC, Renilla luciferase; siRNA, small interfering RNA; stuORF, upstream open reading frame with a strong initiation context; SV40, simian virus 40; TEV, tobacco etch virus; WT, wild-type

Fig 3. Binding of the DENR–MCTS1 complex to tRNA is required for function.

(A) Comparison of MCTS1 with a TGT (PDB-ID: 1J2B). The TGT (light blue, only one molecule from dimer shown) also contains a PUF1947 and PUA domain that directly interact with the tRNA (ribbon, orange). Although the C-terminal MCTS1 domain superimposes well, the N-terminal PUF1947 domains exhibit differences. MCTS1-Phe104 and TGT-Phe519 stack against the last base in the acceptor stem, thereby “measuring” the length of the tRNA. At position of MCTS1-Ala109, TGT contains a lysine residue. (B-B') MCTS1 mutations F104D and A109D strongly impair tRNA binding, assayed by gel shift assay representative of 4 biological replicates). (C) MCTS1 mutations F104D and A109D do not impair binding to DENR. FLAG-tagged MCTS1 variants were IP from HeLa cells, and coimmunoprecipitating endogenous DENR was detected by immunoblot (representative of 2 biological replicates, except the mild drop in DENR binding by the A109D mutation, which is not representative—see S4A Fig). (D) MCTS1 mutation A109L impairs tRNA binding, assayed by gel shift assay (representative of 3 biological replicates). (E) The MCTS1 F104D, A109D, and A109L mutations impair the ability of the DENR–MCTS1 complex to promote translation reinitiation, whereas the F104A mutation does not. Activity of MCTS1 mutants is assayed by reconstituting MCTS1-knockdown HeLa cells with mutated MCTS1 overexpression constructs. Overexpression constructs also contain synonymous substitutions to avoid siRNA-mediated knockdown. Activity is assayed as the ability to promote translation reinitiation downstream of a stuORF as previously reported in [10]. (n = 4). Underlying data available in S1 Data. DENR, density-regulated reinitiation and release factor; FLuc, firefly luciferase; IP, immunoprecipitated; MCTS1, multiple copies in T-cell lymphoma-1; PUA, pseudouridine synthase and archaeosine transglycosylase; PUF1947; RLuc, Renilla luciferase; siRNA, small interfering RNA; stuORF, upstream open reading frame with a strong initiation context; TGT, tRNA-guanine transglycosylase; WT, wild type.

Fig 4. Putative model for the role of DENR–MCTS1 in translation initiation.

DENR–MCTS1 binds tRNAi and subsequently recruits it to the 40S ribosomal subunit for reinitiation. DENR, density-regulated reinitiation and release factor; MCTS1, multiple copies in T-cell lymphoma-1; tRNAi, tRNA interference.