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. 2018 Jun 11;16(6):e2005160. doi: 10.1371/journal.pbio.2005160

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

Fig 3

(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.