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. 2013 Oct;41(18):8615-27.
doi: 10.1093/nar/gkt634. Epub 2013 Jul 22.

Target recognition, RNA methylation activity and transcriptional regulation of the Dictyostelium discoideum Dnmt2-homologue (DnmA)

Sara Müller  1 , Indra M Windhof, Vladimir Maximov, Tomasz Jurkowski, Albert Jeltsch, Konrad U Förstner, Cynthia M Sharma, Ralph Gräf, Wolfgang Nellen
Affiliations

Target recognition, RNA methylation activity and transcriptional regulation of the Dictyostelium discoideum Dnmt2-homologue (DnmA)

Sara Müller et al. Nucleic Acids Res. 2013 Oct.

Abstract

Although the DNA methyltransferase 2 family is highly conserved during evolution and recent reports suggested a dual specificity with stronger activity on transfer RNA (tRNA) than DNA substrates, the biological function is still obscure. We show that the Dictyostelium discoideum Dnmt2-homologue DnmA is an active tRNA methyltransferase that modifies C38 in tRNA(Asp(GUC)) in vitro and in vivo. By an ultraviolet-crosslinking and immunoprecipitation approach, we identified further DnmA targets. This revealed specific tRNA fragments bound by the enzyme and identified tRNA(Glu(CUC/UUC)) and tRNA(Gly(GCC)) as new but weaker substrates for both human Dnmt2 and DnmA in vitro but apparently not in vivo. Dnmt2 enzymes form transient covalent complexes with their substrates. The dynamics of complex formation and complex resolution reflect methylation efficiency in vitro. Quantitative PCR analyses revealed alterations in dnmA expression during development, cell cycle and in response to temperature stress. However, dnmA expression only partially correlated with tRNA methylation in vivo. Strikingly, dnmA expression in the laboratory strain AX2 was significantly lower than in the NC4 parent strain. As expression levels and binding of DnmA to a target in vivo are apparently not necessarily accompanied by methylation, we propose an additional biological function of DnmA apart from methylation.

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Figures

Figure 1.
Figure 1.
In vitro methylation of tRNAAsp(GUC). (A) In vitro methylation of tRNAAsp(GUC) by DnmA at 2 mM, 5 mM and 10 mM MgCl2 and by hDnmt2 at 5 mM Mg2+. The upper panel shows ethidium bromide staining of the in vitro transcripts separated in a denaturing polyacrylamide gel, the lower panel shows incorporated 3H-Me in the tRNAs. Reactions were run for the times indicated. (B) In vivo methylation of cytosines in tRNAAsp(GUC) from different D. discoideum strains. Results of the RNA bisulfite sequencing (454 pyrosequencing) are given in percentage of reads. Numbers of sequence reads are shown in brackets. C49 is methylated by a different methyltransferase and thus serves as an internal standard. All 22 tRNAAsp(GUC) genes result in the same transcript, and no isoacceptors are encoded in the D. discoideum genome. (C) In vitro methylation of small enriched RNA of a dnmAKO strain (ex vivo methylation). The methylation reaction was done for the times indicated.
Figure 2.
Figure 2.
Target site sequences of tRNAs used for in vitro methylation. The position of the putative signature nucleotides are indicated and printed in bold if present. The anticodon is underlined. tRNAGlu(CUC) (not shown) also harbours the nucleotide pattern. Except for the anticodon, both tRNAGlu are identical in the anticodon stem-loop region (Supplementary Figure S2). Only a few differences appear in the acceptor stem.
Figure 3.
Figure 3.
Ex vivo methylation and blocking assay. (A) Methylation of in vitro transcribed tRNAAsp(GUC) was completely blocked when a complementary antisense oligo was hybridized. (B) Using enriched small RNA from dnmAKO cells, ex vivo methylation in the tRNA size class was differentially lost when antisense oligos to tRNAAsp(GUC) and tRNAGlu(UUC) were hybridized before the methylation reaction. The oligo against tRNAGlu(UUC) also covers tRNAGlu(CUC) with minor mismatches (see Supplementary Figure S2). Even with both oligos, a significant amount of 3H incorporation still remained. The upper panel shows the ethidiumbromide stained gel to demonstrate equal loading, the lower panel shows the fluorogram. The arrow marks a band that was specifically lost when the anti tRNAGlu(UUC) oligo was used.
Figure 4.
Figure 4.
C38 is the target for in vitro methylation of tRNAAsp(GUC) and tRNAGlu(UUC). In vitro methylation of tRNAAsp(GUC), tRNAGlu(UUC) and a suppressor tRNAGlu(CUA). C38A mutations confirmed C38 as the target nucleotide. tRNAAsp(GUC) methylation was most efficient.
Figure 5.
Figure 5.
Formation and turnover of tRNA-methyltransferase complexes. Turnover of covalent complexes of tRNA substrates with DnmA and hDnmt2. (A) Examples of time courses on covalent complex formation with tRNAAsp(GUC) and tRNAGlu(UUC). (B) Complex bands in A were quantified and presented on a time scale.
Figure 6.
Figure 6.
tRNA and tRNA fragments associated with DnmA-GFP. After UV crosslinking, RNA bound to DnmA was co-immunoprecipitated under denaturing conditions (CLIP). The number of normalised reads for tRNAs detected by Illumina sequencing is shown for DnmA CLIP and for the control CLIP with GFP. In addition to full-length tRNAs, four classes of tRNA fragments were found, and these are indicated by different shading of the bars. Size and localization of fragments is shown schematically on the simplified clover leaf structure. tRNAs that are not significantly enriched with DnmA are shown in Supplementary Figure S6. In case of multiple gene copys resulting in the same RNA transcript, the sequencing reads were mapped to the first copy in the genome starting from chromsome 1. Only this copy is listed. Sequences and detailed information on gene copies and potential isoacceptors are listed in Supplementary Table S2.
Figure 7.
Figure 7.
In vitro methylation of tRNAGly(GCC) by recombinant DnmA and hDnmt2. In vitro transcribed tRNAs were methylated in vitro as described before. The top panel shows the ethidium-bromide stained gel, the bottom panel the fluorogram of 3H-labelled methylated tRNAs. As a control tRNAAsp(GUC) methylation is shown.
Figure 8.
Figure 8.
Expression, localization and tRNAAsp(GUC) methylation activity of DnmA under various conditions. (A) dnmA expression is regulated during the cell cycle. Cells were arrested in the cell cycle by cold treatment and then released by transfer to 22°C. Cells were counted every 30 min (grey dots and grey line), and samples were taken for qPCR (black bars). After cell division, dnmA expression increased ∼5-fold and then rapidly declined to basal levels. Normalization was done on vegetative growing cells. dnmA expression is only shown from 3 to 4.5 h during recovery. (B) DnmA is lost from the nucleus during mitosis (arrow). The three cells on the right are in S-phase as indicated by the RFP-PCNA marker and accumulate DnmA in the nucleus (for further details see movie in Supplementary Material). (C) Relative quantification of dnmA expression levels in AX2 cells after 2.5 h recovery from cold shock at 4°C. Expression of dnmA increased >40-fold and returned within 30 min to basal levels (n = 3). (D) At 16 h of development, dnmA expression increased ∼46-fold in the NC4 strain. In AX2 cells, expression increased only ∼5-fold. (E) At 16 h of development, in vivo methylation of C38 in tRNAAsp(GUC) increased up to 75% in the D. discoideum strain NC4, whereas no significant increase was observed in AX2 cells.
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