Identification of the enzyme responsible for N1-methylation of pseudouridine 54 in archaeal tRNAs

Abstract

tRNAs from all three kingdoms of life contain a variety of modified nucleotides required for their stability, proper folding, and accurate decoding. One prominent example is the eponymous ribothymidine (rT) modification at position 54 in the T-arm of eukaryotic and bacterial tRNAs. In contrast, in most archaea this position is occupied by another hypermodified nucleotide: the isosteric N1-methylated pseudouridine. While the enzyme catalyzing pseudouridine formation at this position is known, the pseudouridine N1-specific methyltransferase responsible for this modification has not yet been experimentally identified. Here, we present biochemical and genetic evidence that the two homologous proteins, Mja_1640 (COG 1901, Pfam DUF358) and Hvo_1989 (Pfam DUF358) from Methanocaldococcus jannaschii and Haloferax volcanii, respectively, are representatives of the methyltransferase responsible for this modification. However, the in-frame deletion of the pseudouridine N1-methyltransferase gene in H. volcanii did not result in a discernable phenotype in line with similar observations for knockouts of other T-arm methylating enzymes.

FIGURE 1.

MjNep1 mis-methylates pseudouridine containing substrates corresponding to tRNA T-arms at position 55. (A) Chemical structure of N1-methyl pseudouridine. (B) Secondary structure and sequences of minimal substrates for MjNep1 derived from the small subunit rRNA helix 35 (Wurm et al. 2010). Nucleotides that are similar between these substrates and T-arm sequences of archaeal tRNAs are highlighted in red. (C) Secondary structure of H. volcanii tRNA^Trp with the modified nucleotide positions indicated and sequence similarities to MjNep1 substrates highlighted in red. The T-arm sequences and secondary structures of H. volcanii and M. jannaschii tRNA^Trp are virtually identical. (D) Methylation activity of MjNep1 toward 17 mer RNA substrates resembling the T-arm of M. jannaschii tRNA^Trp with pseudoruridines at position 54 (top), 55 (middle), or both (bottom) analyzed by MALDI mass spectrometry. Only the RNAs with pseudouridine at positions 55 or 54 and 55 are methylated after 5-min reaction time at 65°C. (E) Identification of the methylation site in the RNA substrate containing two pseudouridine residues at positions corresponding to Ψ54 and Ψ55 in full-length tRNAs by partial acidic hydrolysis coupled to MALDI mass spectrometry of the reaction product. Shown are the sections of the spectrum for two indicated 5′ fragments.

FIGURE 2.

Structural similarity between Nep1 and Mja_1640. (A) Overlay of the structures of the MjNep1 (pdb 3bbe) and Mja_1640 (pdb 3aia) dimers revealing the similar overall fold typical for SPOUT-class RNA methyltransferases. (B) Model of pseudouridine bound to the active center of Nep1 derived from the structure of yeast Nep1 bound to S-adenosylhomocysteine and an inhibitor RNA containing uridine instead of pseudouridine at the reactive position (pdb 3oin). The amino acids R88, D90, and A234, which form hydrogen bonds to the pseudouridine carbonyl groups and orient the N1 nitrogen toward the sulfur atom of the cofactor, are colored by atom types. The equivalent amino acids in Mja_1640 are shown in gray and numbered according to the Mja_1640 numbering scheme.

FIGURE 3.

RNA-methyltransferase activity of Mja_1640. (A) Methylation activity of Mja_1640 toward T-arm substrates containing no pseudouridine (top), pseudouridine at position 55 (second from top), pseudouridine at position 54 (second from bottom), or pseudouridine at positions 54 and 55 (bottom). In the last two cases a complete methylation is observed after a 5-min reaction time as indicated by the shift in the mass spectrum by 14 Da. (B) Identity of the modified nucleotide. HPLC chromatograms of nucleoside mixtures after digestion and dephosphorylation of the 17 mer RNA substrate containing pseudouridine at position 54. The HPLC chromatogram of the unreacted substrate RNA contains only peaks corresponding to Ψ, C, U, G, and A (red trace), whereas upon methylation by Mja_1640 the Ψ peak disappears, and a novel peak appears with a retention time typical for N1-methyl pseudouridine (black trace). A reference for commercially obtained N1-methyl pseudouridine is shown on top. (C) Mass spectra of the T-arm substrate containing two pseudouridines after methylation with Mja_1640 and limited acidic hydrolysis. Shown are the sections of the spectrum for two indicated 5′- fragments indicative of methylation at the pseudouridine corresponding to position 54 in full-length tRNAs. (D) Secondary structure for a 74 mer RNA substrate resembling unmodified M. jannaschii tRNA^Trp with a single pseudoruridine at position 54. HPLC traces for unreacted (red) and Mja_1640-treated 74 mer RNA after digestion and dephosphorylation and after a 1-h reaction time (black). A novel peak corresponding to N1-methyl pseudouridine appears following incubation with Mja_1640 and SAM.

FIGURE 4.

Hvo_1989 activity in vivo. (A) Hvo_1989 and Mja_1640 are highly homologous proteins, as seen from the sequence alignment of members of the DUF 358 family. Identical amino acids of the archaeal members of this family are colored in red, similar amino acids are shown in green. The arginine and the aspartate important for pseudouridine recognition and orientation are indicated by a star. Only in the bacterial member of this family these amino acids are a histidine and a glutamate, respectively. (B) Nucleoside analysis of unfractionated tRNAs isolated from wild-type (black) and the in-frame deletion mutant cells (red) and mutant cells complemented with the pSD1/M2-18 plasmid (green). For the tRNAs from wild-type and complemented strains the N1-methyl pseudouridine signal (indicated in red) is clearly present, while the signal is absent in the case of the in-frame deletion mutant. The putative assignments for the signals corresponding to other modified nucleosides are also indicated (Edmonds et al. 1991). (C) Nucleoside analysis of tRNAs isolated from in-frame deletion mutant cells (red) and the same tRNAs after incubation with SAM and Mja_1640 (black). The N1-methyl pseudouridine signal only appears after incubation with Mja_1640.