Identification of human tRNA:m5C methyltransferase catalysing intron-dependent m5C formation in the first position of the anticodon of the pre-tRNA Leu (CAA)

Abstract

We identified a human orthologue of tRNA:m5C methyltransferase from Saccharomyces cerevisiae, which has been previously shown to catalyse the specific modification of C34 in the intron-containing yeast pre-tRNA Leu (CAA). Using transcripts of intron-less and intron-containing human pre-tRNA Leu (CAA) genes as substrates, we have shown that m5C34 is introduced only in the intron-containing tRNA precursors when the substrates were incubated in the HeLa extract. m5C34 formation depends on the nucleotide sequence surrounding the wobble cytidine and on the structure of the prolongated anticodon stem. Expression of the human Trm4 (hTrm4) cDNA in yeast partially complements the lack of the endogenous Trm4p enzyme. The yeast extract prepared from the strain deprived of the endogenous TRM4 gene and transformed with hTrm4 cDNA exhibits the same activity and substrate specificity toward human pre-tRNALeu transcripts as the HeLa extract. The hTrm4 MTase has a much narrower specificity against the yeast substrates than its yeast orthologue: human enzyme is not able to form m5C at positions 48 and 49 of human and yeast tRNA precursors. To our knowledge, this is the first report showing intron-dependent methylation of humanpre-tRNA Leu (CAA) and identification of human gene encoding tRNA methylase responsible for this reaction.

Figure 1

Cloverleaf structure of ${\text{tRNA}}_{\left(\text{CAA}\right)}^{\text{Leu}}$ precursor containing intron (a), mature tRNA (b) and two mutants with partially deleted introns (derived from plasmids pHLIVS2 ΔI1, pHLIVS2 ΔI2) (c and d). Anticodon sequence is underlined, arrows point to splice sites.

Figure 2

Analysis of m⁵C₃₄ content in intron-less (a) and intron-containing (b) human ${\text{pre-tRNA}}_{\left(\text{CAA}\right)}^{\text{Leu}}$ by two-dimensional thin layer chromatography. Pre-tRNAs were incubated in the cell-free nuclear HeLa extract. tRNA^Leu precursors were synthesized and treated as described in Materials and Methods.

Figure 3

Detection of m⁵C in bulk low-molecular weight RNA extracted from yeast wild-type strain BY4742 (b), BY4742 ΔTRM4 (c), BY4742 transformed with pYES2 containing yeast Trm4p cDNA (d) and BY4742 ΔTRM4 transformed with pYES2 containing hTrm4 cDNA (e). Separation of a test nucleoside mixture (Ψ, U, C, m⁵C, G and A) is presented in (a). The total RNA fraction was completely hydrolysed by nuclease P1 and the resulting nucleotides dephosphorylated by CIAP. Analysis of nucleosides was performed as described in Materials and Methods.

Figure 4

Analysis of m⁵C₃₄ content in human ${\text{pre-tRNA}}_{\left(\text{CAA}\right)}^{\text{Leu}}$ without intron and containing intron by 2-D thin layer chromatography. Pre-tRNAs were incubated in yeast S-10 extract derived from wild-type strain (a), BY4742 ΔTRM4 (b), BY4742 ΔTRM4 transformed with pYES2-containing hTrm4 cDNA (c) and BY4742 ΔTRM4 transformed with pYES2-containing yeast Trm4p cDNA (d). (e) Incubation of yeast ${\text{pre-tRNA}}_{\left(\text{CAA}\right)}^{\text{Leu}}$ containing intron in S-10 extract prepared from BY4742 ΔTRM4 transformed with pYES2-containing hTrm4 cDNA. tRNA^Leu precursors were synthesized and treated as described in Materials and Methods.

Figure 5

Kinetics of m⁵C₃₄ formation in human intron-containing ${\text{pre-tRNA}}_{\left(\text{CAA}\right)}^{\text{Leu}}$. ³²P-labelled pre-tRNA^Leu transcripts were incubated with S-10 yeast extract prepared from BY4742 Δtrm4 + hTrm4 cDNA at 30°C for 10, 20, 40 and 90 min and analyzed for the relative amount of m⁵C residues at position 34. The measurements were taken as described in Materials and Methods.

Figure 6

Analysis of m⁵C₄₈ (a–c) and m⁵C₄₉ (d) content in human and yeast tRNAs using hTrm4 and yeast Trm4p enzymes by 2D TLC. Pre-tRNAs were labelled using [α-³²P] GTP during T7 RNA polymerase transcription, gel purified and incubated in S-10 extract. (a) BY4742 Δtrm4 + hTrm4 yeast strain and human pre-tRNA^Leu, (b) BY4742 Δtrm4 + yeast Trm4p and human pre-tRNA^Leu, (c) BY4742 Δtrm4 + hTrm4 (left) and BY4742 Δtrm4 + Trm4p (right) and yeast ${\text{tRNA}}_{\left(\text{AGA}\right)}^{\text{Ser}}$, and (d) BY4742 Δtrm4 + hTrm4 (left), BY4742 Δtrm4 + Trm4p (right) and yeast ${\text{tRNA}}_{\left(\text{GUC}\right)}^{\text{Asp}}$. Yeast ${\text{tRNA}}_{\left(\text{GUC}\right)}^{\text{Asp}}$ and ${\text{tRNA}}_{\left(\text{AGA}\right)}^{\text{Ser}}$ genes do not contain introns.