Human DNMT2 methylates tRNA(Asp) molecules using a DNA methyltransferase-like catalytic mechanism

Abstract

Although their amino acid sequences and structure closely resemble DNA methyltransferases, Dnmt2 proteins were recently shown by Goll and colleagues to function as RNA methyltransferases transferring a methyl group to the C5 position of C38 in tRNA(Asp). We observe that human DNMT2 methylates tRNA isolated from Dnmt2 knock-out Drosophila melanogaster and Dictyostelium discoideum. RNA extracted from wild type D. melanogaster was methylated to a lower degree, but in the case of Dictyostelium, there was no difference in the methylation of RNA isolated from wild-type and Dnmt2 knock-out strains. Methylation of in vitro transcribed tRNA(Asp) confirms it to be a target of DNMT2. Using site directed mutagenesis, we show here that the enzyme has a DNA methyltransferase-like mechanism, because similar residues from motifs IV, VI, and VIII are involved in catalysis as identified in DNA methyltransferases. In addition, exchange of C292, which is located in a CFT motif conserved among Dnmt2 proteins, strongly reduced the catalytic activity of DNMT2. Dnmt2 represents the first example of an RNA methyltransferase using a DNA methyltransferase type of mechanism.

FIGURE 1.

Alignment of Dnmt2 family. (A) Multiple sequence alignment of Dnmt2 proteins from human, mouse, D. melanogaster, E. histolytica, D. discoideum, and S. pombe. The regions corresponding to catalytic motifs of DNA MTases are labeled. The residues selected for analysis are marked with arrows and labeled. (B) Structure of human DNMT2 protein (Dong et al. 2001). The residues studied in this work are shown in space fill representation. Note that the loop containing C79A is not ordered in the structure, such that this residue is not visible. (C) Schematic picture of the catalytic mechanisms proposed for DNA MTases and RNA MTases. For details see the text (data adapted from Bujnicki et al. 2004).

FIGURE 2.

Protein purification and circular dichroism spectra of the DNMT2 wild type and all variants. (A) Coomassie stained SDS polyacrylamide gel showing the purified wild-type and mutant DNMT2 proteins. (B) Far UV circular dichroism spectra of DNMT2 and its variants recorded using 10 μM enzyme in 10 mM Tris/HCl (pH 7.5), 200 mM KCl solution. The figure shows a superposition of the spectra measured with the wild-type and mutant proteins.

FIGURE 3.

RNA binding by DNMT2 and its variants analyzed by the nitrocellulose filter binding assay. (A) Dot blot analysis of the wild-type DNMT2 binding the ³²P labeled in vitro transcribed tRNA^Asp. (B) Binding curves of DNMT2 wild type and C79A and E119A mutants to in vitro transcribed and ³²P-labeled tRNA^Asp. Diamonds denote E119A mutant, triangles wild-type DNMT2, circles C79A. The experimental data points were fitted to a bimolecular binding equilibrium to determine the K_Ass values.

FIGURE 4.

Generation of a dDnmt2 knock-out Drosophila melanogaster strain. (A) Schematic drawing of the structure of the dDnmt2¹⁴⁹ null allele. A null allele of dDnmt2 was generated after remobilization of the P element GE15695 inserted 128 bp upstream of the dDnmt2 gene. dDnmt2¹⁴⁹ shows an insertion of 59 bp of 5′ P element sequences 9 bp downstream of first ATG position within the Dnmt2 ORF introducing an early stop codon. (B) RT-PCR detection of dDnmt2 mRNA. No specific transcript is detected in dDnmt2¹⁴⁹ null embryos. Lamine mRNA was used as internal control.

FIGURE 5.

Methylation of total RNA extracted from the dnmt2 knock-out D. melanogaster cells by wild-type DNMT2 and its variants. (A) Human DNMT2 methylates RNA molecules in the size range of tRNA. A DNA molecular weight marker was used and the approximate positions of the DNA fragments of different lengths (in nucleotides) are indicated. (B) Quantification of DNMT2 catalyzed methyl group transfer to RNA. In this experiment, DNA methylated by M.SssI was used to calibrate the RNA methylation by DNMT2. Different amounts of methylated DNA were loaded into the different lanes of the gel. The amount of methyl groups in each lane (in picomoles) is indicated above the corresponding well. In this experiment, 10 μg of total RNA were loaded into each well.

FIGURE 6.

In vitro methylation of RNA by DNMT2 and its variants. (A) Time course of RNA methylation by wild-type DNMT2. The upper panel shows the time course of incorporation of radioactivity into RNA. The radioactive bands were analyzed quantitatively and the data fit to a single exponential reaction progress curve as shown in the lower panel. (B) Example of RNA methylation by the DNMT2 variants after 3 h reaction time.

FIGURE 7.

In vitro methylation of different RNA substrates by DNMT2. (A) In vitro transcribed tRNA^Asp can be methylated by DNMT2 wild-type protein. A DNA molecular weight marker was used and the approximate positions of the DNA fragments of different lengths (in nucleotides) are indicated. (B) Methylation of the D. discoideum total RNA extract from the Ax2 wild-type cells and the dnmA knock-out cells.