THUMPD3–TRMT112 is a m2G methyltransferase working on a broad range of tRNA substrates

Abstract

Post-transcriptional modifications affect tRNA biology and are closely associated with human diseases. However, progress on the functional analysis of tRNA modifications in metazoans has been slow because of the difficulty in identifying modifying enzymes. For example, the biogenesis and function of the prevalent N2-methylguanosine (m 2 G) at the sixth position of tRNAs in eukaryotes has long remained enigmatic. Herein, using a reverse genetics approach coupled with RNA-mass spectrometry, we identified that THUMP domain-containing protein 3 (THUMPD3) is responsible for tRNA: m 2 G6 formation in human cells. However, THUMPD3 alone could not modify tRNAs. Instead, multifunctional methyltransferase subunit TRM112-like protein (TRMT112) interacts with THUMPD3 to activate its methyltransferase activity. In the in vitro enzymatic assay system, THUMPD3–TRMT112 could methylate all the 26 tested G6-containing human cytoplasmic tRNAs by recognizing the characteristic 3′-CCA of mature tRNAs. We also showed that m 2 G7 of tRNA Trp was introduced by THUMPD3–TRMT112. Furthermore, THUMPD3 is widely expressed in mouse tissues, with an extremely high level in the testis. THUMPD3 -knockout cells exhibited impaired global protein synthesis and reduced growth. Our data highlight the significance of the tRNA: m 2 G6/7 modification and pave a way for further studies of the role of m 2 G in sperm tRNA derived fragments.

Required Elements in tRNA for Methylation by the Eukaryotic tRNA (Guanine-N2-) Methyltransferase (Trm11-Trm112 Complex)

“…In the study, we reported that the THUMP domain in T. kodakarensis Trm11 recognizes the CCA terminus in tRNA [ 8 ]. The recognition of the 3′-end of substrate tRNA by a THUMP domain has also been reported for other tRNA modification enzymes [ 8 , 9 , 10 , 19 , 20 ]. For example, the crystal structure of complex of Thermotoga maritima ThiI (tRNA 4-thiouridine synthetase) and truncated tRNA revealed that the THUMP domain in ThiI captures the 3′-terminal region in tRNA [ 20 ].…”

“…Trm11 is a typical Rossmann fold S-adenosyl-L-methionine (AdoMet)-dependent methyltransferase (COG 1041) with a thiouridine synthase, methyltransferase and pseudouridine synthase (THUMP) domain fused to its N-terminal region [ 4 , 5 , 8 , 9 , 10 ]. The THUMP domain is frequently observed in tRNA modification enzymes [ 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 ] and is often required for binding to the CCA terminal region of tRNA [ 8 , 9 , 10 , 19 , 20 ]. In the Trm11-Trm112 complex, Trm112 activates the tRNA methyltransferase activity of Trm11 [ 4 , 9 ].…”

“…It should be mentioned that Trm112 activates not only Trm11 but also other methyltransferases in yeast (for example, Trm9 [ 21 ], Bud23 [ 22 , 23 ] and Mtq2 [ 6 , 24 , 25 , 26 ]). Furthermore, a human ortholog of Trm112, TRMT112, interacts with at least seven human methyltransferases (WBSCR22 (responsible for the formation of 7-methylguanosine at position 1636 in 18S rRNA) [ 27 ], METTL5 (responsible for the formation of N 6 -methyladenosine at position 1832 in 18S rRNA) [ 28 ], HEMK2 (responsible for the methylation of a glutamine side chain of eRF1 protein) [ 29 ], ALKBH8 (responsible for 5-methoxycarbonylmethyluridine derivatives at position 34 in tRNA) [ 30 , 31 , 32 , 33 ], TRMT11 (probably responsible for m 2 G10 in tRNA, although the enzymatic activity has not yet been confirmed experimentally) [ 4 , 34 ], THUMPD2 (function unknown) [ 34 ] and THUMPD3 (responsible for the production of m 2 G6 and m 2 G7 in tRNA)) [ 19 ]. Thus, in eukaryotes, Trm112 acts as a hub-protein, which regulates tRNA, rRNA and protein methylation [ 6 , 34 ].…”

Human TRMT112-Methyltransferase Network Consists of Seven Partners Interacting with a Common Co-Factor

“…We performed three biological replicates and, in all cases, seven MTases with H/L ratios ranging from 5 to 24 were pulled down (Figure 1D). Among these proteins were the already known TRMT112 interaction partners WBSCR22 [9,13], N6AMT1 [20], ALKBH8 [26], METTL5 [10] and THUMPD3 [29], but also novel, uncharacterised proteins THUMPD2 and TRMT11. THUMPD2 is specific to mammalian cells, but TRMT11 homologue in yeast Trm11 is a tRNA MTase [30].…”

“…THUMPD3, together with TRMT112, is a m 2 G methyltransferase working on a broad range of tRNA substrates. THUMPD3-knockout cells exhibit an impaired global protein synthesis and reduced growth [29].…”