New substrates and determinants for tRNA recognition of RNA methyltransferase DNMT2/TRDMT1

Abstract

Methylation is a common post-transcriptional modification of tRNAs, particularly in the anticodon loop region. The cytosine 38 (C38) in tRNAs, such as tRNA^Asp-GUC, tRNA^Gly-GCC, tRNA^Val-AAC, and tRNA^Glu-CUC, can be methylated by human DNMT2/TRDMT1 and some homologs found in bacteria, plants, and animals. However, the substrate properties and recognition mechanism of DNMT2/TRDMT1 remain to be explored. Here, taking into consideration common features of the four known substrate tRNAs, we investigated methylation activities of DNMT2/TRDMT1 on the tRNA^Gly-GCC truncation and point mutants, and conformational changes of mutants. The results demonstrated that human DNMT2/TRDMT1 preferred substrate tRNA^Gly-GCC in vitro. L-shaped conformation of classical tRNA could be favourable for DNMT2/TRDMT1 activity. The complete sequence and structure of tRNA were dispensable for DNMT2/TRDMT1 activity, whereas T-arm was indispensable to this activity. G19, U20, and A21 in D-loop were identified as the important bases for DNMT2/TRDMT1 activity, while G53, C56, A58, and C61 in T-loop were found as the critical bases. The conserved CUXXCAC sequence in the anticodon loop was confirmed to be the most critical determinant, and it could stabilize C38-flipping to promote C38 methylation. Based on these tRNA properties, new substrates, tRNA^Val-CAC and tRNA^Gln-CUG, were discovered in vitro. Moreover, a single nucleotide substitute, U32C, could convert non-substrate tRNA^Ala-AGC into a substrate for DNMT2/TRDMT1. Altogether, our findings imply that DNMT2/TRDMT1 relies on a delicate network involving both the primary sequence and tertiary structure of tRNA for substrate recognition.

Keywords: DNMT2/TRDMT1; conformational change; methylation kinetics; recognition determinant; tRNA.

Figure 1.

Human DNMT2/TRDMT1-tRNA complex structure and DNMT2/TRDMT1 methylation preference for tRNA substrates. A. Tertiary L-shaped nucleotide sequence representation of human tRNA^Gly-GCC predicted via RNAComposer. The tertiary interactions were conservative in all the cytoplasmic tRNAs and highlighted in dashed lines. B. Tertiary L-shaped structure of human tRNA^Gly-GCC presented in a cartoon by PyMOL 1.7.0. Structural domains of tRNA^Gly-GCC were indicated by different colours. C. Human DNMT2/TRDMT1-tRNA complex structure. tRNA^Gly-GCC was docked into the crystal structure of human DNMT2/TRDMT1 (PDB ID: 1G55) by a web server, HDOCK. The protein contact potential of DNMT2/TRDMT1 was calculated by PyMOL 1.7.0. S-adenosyl-L-homocysteine (SAH) and cytosine 38 (C38) were shown in sticks. D. Bar graph of ³H incorporated into different tRNA substrates by DNMT2/TRDMT1 at 70 min. E. Hyperbola plot of methylation activities of DNMT2/TRDMT1 on tRNA^Gly-GCC calculated using Michaelis-Menten equation. Data were expressed as the mean ± SD from three independent measurements

Figure 2.

Sequence conservation of tRNA substrates and tRNA^Gly-GCC mutant structures. A. Sequence alignment of tRNA substrates of human DNMT2/TRDMT1 under ‘default’ mode by ClustalW multiple sequence alignment program in CLUSTAL X (2.0.12) software. B. Cloverleaf structures of tRNA^Gly-GCC mutants predicted by tRNAscan-SE 2.0

Figure 3.

Methylation and structural characterizations of tRNA^Gly-GCC deletion mutants. A. Bar graph of ³H incorporated into tRNA^Gly-GCC and its deletion mutants by DNMT2/TRDMT1 at 70 min. B. CD spectra of tRNA^Gly-GCC deletion mutants. C. Tertiary structures of tRNA^Gly-GCC deletion mutants predicted. Data were expressed as the mean ± SD from three independent measurements

Figure 4.

Methylation and structural characterizations of tRNA^Gly-GCC D-loop mutants. A. Bar graph of ³H incorporated into tRNA^Gly-GCC mutants in D-loop by DNMT2/TRDMT1 at 70 min. B. CD spectra of tRNA^Gly-GCC D-loop mutants. C. Tertiary structures of tRNA^Gly-GCC D-loop mutants predicted. Data were expressed as the mean ± SD from three independent measurements

Figure 5.

Methylation and structural characterizations of tRNA^Gly-GCC T-loop mutants. A. Bar graph of ³H incorporated into tRNA^Gly-GCC mutants in T-loop by DNMT2/TRDMT1 at 70 min. B. CD spectra of tRNA^Gly-GCC T-loop mutants. C. Tertiary structures of tRNA^Gly-GCC T-loop mutants predicted. Data were expressed as the mean ± SD from three independent measurements

Figure 6.

Methylation and structural characterizations of tRNA^Gly-GCC anticodon loop mutants. A. Bar graph of ³H incorporated into tRNA^Gly-GCC mutants in anticodon loop by DNMT2/TRDMT1 at 70 min. B. CD spectra of tRNA^Gly-GCC anticodon loop mutants. C. Tertiary structures of tRNA^Gly-GCC anticodon loop mutants predicted. Hydrogen bond interactions between C38 and mutational bases were indicated in yellow dashed lines. Data were expressed as the mean ± SD from three independent measurements

Figure 7.

Methylation, sequential and structural characterizations of C38-containing non-substrate tRNAs. A. Bar graph of ³H incorporated into non-substrate tRNAs by DNMT2/TRDMT1 at 70 min. B. Sequence alignment of tRNA^Ala-AGC and four known tRNA substrates of DNMT2/TRDMT1. C. CD spectra of tRNA^Ala-AGC and tRNA^AlaU32G. D. Tertiary structures of tRNA^Ala-AGC and tRNA^AlaU32G predicted. The hydrogen bond interactions between C38 and mutational bases were denoted in yellow dashed lines. E. Sequence alignment of tRNA^Val-CAC, tRNA^Gln-CUG, and four known substrates of DNMT2/TRDMT1. Data were expressed as the mean ± SD from three independent measurements