The archaeal COG1901/DUF358 SPOUT-methyltransferase members, together with pseudouridine synthase Pus10, catalyze the formation of 1-methylpseudouridine at position 54 of tRNA

Abstract

The methylation of pseudouridine (Ψ) at position 54 of tRNA, producing m(1)Ψ, is a hallmark of many archaeal species, but the specific methylase involved in the formation of this modification had yet to be characterized. A comparative genomics analysis had previously identified COG1901 (DUF358), part of the SPOUT superfamily, as a candidate for this missing methylase family. To test this prediction, the COG1901 encoding gene, HVO_1989, was deleted from the Haloferax volcanii genome. Analyses of modified base contents indicated that while m(1)Ψ was present in tRNA extracted from the wild-type strain, it was absent from tRNA extracted from the mutant strain. Expression of the gene encoding COG1901 from Halobacterium sp. NRC-1, VNG1980C, complemented the m(1)Ψ minus phenotype of the ΔHVO_1989 strain. This in vivo validation was extended with in vitro tests. Using the COG1901 recombinant enzyme from Methanocaldococcus jannaschii (Mj1640), purified enzyme Pus10 from M. jannaschii and full-size tRNA transcripts or TΨ-arm (17-mer) fragments as substrates, the sequential pathway of m(1)Ψ54 formation in Archaea was reconstituted. The methylation reaction is AdoMet dependent. The efficiency of the methylase reaction depended on the identity of the residue at position 55 of the TΨ-loop. The presence of Ψ55 allowed the efficient conversion of Ψ54 to m(1)Ψ54, whereas in the presence of C55, the reaction was rather inefficient and no methylation reaction occurred if a purine was present at this position. These results led to renaming the Archaeal COG1901 members as TrmY proteins.

FIGURE 1.

Enzymatic post-transcriptional modifications of selected uridines in RNA. (A) Schematic consensus of tRNA secondary structures indicating the U54 target within the highly conserved 7-nt TΨ-loop. The dashed line indicates a reverse Hoogsteen base pair within the loop. In the majority of Archaea, U54 is first isomerized into Ψ54 by a tRNA pseudouridine synthase, aPus10 (symbol “a” preceding the acronym of the enzyme denotes “archaeal,” “b” means “bacterial,” and “e” means “eukaryal”). In certain Archaea, Ψ54 can be further methylated into m¹Ψ54 by a SPOUT-type, SAM-dependent methyltransferase designated TrmY (this work). In Thermococcales, Bacteria, and Eukarya, U54 is methylated into m⁵U54 (riboT) by a distinct SAM-dependent tRNA-U54 methyltransferase aTrmU54, bTrmA, and eTrm2p, respectively. In thermophilic organisms, m⁵U54 can be further thiolated into s²m⁵U (s²T) by the heteromeric enzyme TtuA/TtuB. (B) Schematic consensus of a portion of 16S rRNA as part of domain IV, encompassing the highly conserved 8-nt helix 35 Ψ-loop. U914 in 16S rRNA of M. jannaschii, corresponding to U1191 in 18S rRNA of S. cerevisiae, is first isomerized into Ψ by an archaeal enzyme (or enzymatic system) that remains to be identified. In S. cerevisiae this reaction is mediated by the snoRNP complex consisting of the pseudouridine synthase eCbf5 and snR35 guide RNA. The methylation of N1-atom of the uracil ring is further catalyzed by a SPOUT-type and SAM-dependent methyltransferase, Nep1 (also referred as Emg1). Only in some Eukarya (such as yeast, Drosophila, HeLa cells), is the m¹Ψ further hypermodified into a acp³m¹Ψ derivative. On the right part of the figure are the different uridine derivatives. The dashed lines through the structure of m¹Ψ and acp³m¹Ψ show the axis of base rotation during the isomerization process. The asterisks with small arrows indicate the atoms normally engaged in the reverse Hoogsteen base pair with A58.

FIGURE 2.

Construction and phenotype of the trmY-deleted strain of H. volcanii. (A) Deletion of the trmY gene was confirmed by PCR; (left) PCR products using primers designed to anneal outside the gene (HvO_1989_Ext_F and HvO_1989_Ext_R) confirm a genomic rearrangement of correctly predicted sizes in WT and mutant strains; (middle) PCR products using primers designed to anneal within the target gene (HvO_1989_Int_F and HvO_1989_Int_R) show the absence of the trmY internal segment in the mutant and its presence in the WT strain; (right). To confirm the presence of VNG_1980c in trans, primers were designed to anneal to the complementing gene (HsaI_COG1901_Fwd and HsaI_COG1901_Rev). The predicted size of the fragment is observed in the rescued strain. (B) LC-MS/MS analysis of tRNA extracted from H26 (wild-type) and VDC2376 (ΔtrmY) showing the UV trace at 254nm (top). The 259 m/z ion that corresponds to the protonated molecular weight (MH⁺) of m¹Ψ was detected by MS at 13.36 min in the WT background, while no 259 m/z ion was detected in the ΔtrmY strain (bottom).

FIGURE 3.

Formation of m¹Ψ of H. volcanii tRNA is mediated by TrmY. (A) Nuclease P1 digests of uniformly labeled tRNAs were resolved by 2D-TLC. pA, pC, pG, pU, pΨ, and pm¹Ψ indicate 5′-phosphorylated A, C, G, U, Ψ, and m¹Ψ, respectively. The radioactive spot corresponding to pm¹Ψ is present in both wild-type (H26) and complemented (ΔtrmY+pHTrmY) strains, but is absent from ΔtrmY strain (middle). (B) CMCT-primer extension analyses to determine the modification status of residue at position 54 of H. volcanii elongator tRNA^Met were done using primer Met-CCA2 (position marked in Fig. 4A) and total small RNA of wild-type, ΔtrmY, and ΔtrmY+pHTrmY strains. RNAs were treated with (+) or without (−) CMCT for the indicated time (in minutes), followed by alkali (OH-) treatment (+) or no treatment (−). Positions of tRNA residues 54 and 55 are marked on the side. A dark band in CMCT followed by alkali treatment lanes, with an increased intensity in the 20-min treatment lane, indicates the presence of Ψ at that position. These reactions show that unmethylated Ψs are present at position 55 in all three strains, but at position 54 only in the ΔtrmY strain (the band is marked by an arrow).

FIGURE 4.

M. jannaschii TrmY can convert Ψ54 of tRNA to m¹Ψ54. (A) Sequences of transcripts used in the reactions. Mutations changing U55 of tRNA^Trp and U54 of tRNA^Met are indicated. The arrow in tRNAMET sequence indicates the position of primer Met-CCA2 used for CMCT-primer extension analyses shown in Figure 3B. (B,C) [α-³²P]UTP-labeled tRNA^Trp was first pseudouridylated by M. jannaschii Pus10 (Mj-Pus10) and then methylated by M. jannaschii TrmY (Mj-TrmY) as described in Materials and Methods. Nuclease P1 or RNase T2 (indicated in panels) digests of purified products were resolved by 2D-TLC on cellulose plates. “p” before or after a nucleoside letter indicates the 5′ or 3′ phosphate of that nucleoside. (Pi) inorganic phosphate. (D) Treatments similar to those in B using [α-³²P]UTP-labeled tRNA^Met as substrate. The middle panel here (and in B) is shown to indicate that Ψ in the tRNA is produced by Mj-Pus10. (E) TLC separation of RNase T2 digest of [α-³²P]CTP-labeled tRNA^Trp following treatment with Mj-TrmY. (F–I) TLC separation of nuclease P1 digests of [α-³²P]UTP-labeled T-arm-Trp, mutant tRNA^Trp-U55A, tRNA^Trp-U55G, and tRNA^Trp-U55C, respectively (indicated in the panels), following treatment with Mj-TrmY.

FIGURE 5.

Phylogeny of the COG1901 family and structural comparison of Mj1640 dimer with VV2_1434 dimer. (A) Unrooted Bayesian tree of 42 bacterial and 72 archaeal proteins identified as COG1901 in GenBank and are listed in Supplemental file 1. For clarity, only taxa from major clades are labeled. The scale bar indicates the average number of substitutions per site. Numbers at branches represent posterior probabilities as inferred by Mr. Bayes; for clarity, only major branches are labeled. The division between Bacteria and Archaea is well supported. (*) A. borkumensis SK2; (+) S. baltica OS155, OS183, and OS185. (B) Ribbon representation of Mj1640 dimer (left); Electrostatic surface potential presentation of Mj1640 dimer (right), with blue and red colors corresponding to positively and negatively charged patches, respectively. (C) Ribbon (left) and electrostatic surface potential representation (right) of VV2_1434 dimer.