A novel enzymatic pathway leading to 1-methylinosine modification inHaloferax volcaniitRNA

Abstract

Transfer RNAs of the extreme halophile Haloferax volcanii contain several modified nucleosides, among them 1-methylpseudouridine (m1 psi), pseudouridine (psi), 2'-0-methylcytosine (Cm) and 1-methylinosine (m1l), present in positions 54, 55, 56 and 57 of the psi-loop, respectively. At the same positions in tRNAs from eubacteria and eukaryotes, ribothymidine (T-54), pseudouridine (psi-55), non-modified cytosine (C-56) and non-modified adenosine or guanosine (A-57 or G-57) are found in the so-called T psi-loop. Using as substrate a T7 transcript of Haloferax volcanii tRNA(Ile) devoid of modified nucleosides, the enzymatic activities of several tRNA modification enzymes, including those for m1 psi-54, psi-55, Cm-56 and m1l-57, were detected in cell extracts of H.volcanii. Here, we demonstrate that modification of A-57 into m1l-57 in H.volcanii tRNA(Ile) occurs via a two-step enzymatic process. The first step corresponds to the formation of m1A-57 catalyzed by a S-adenosylmethionine-dependent tRNA methyltransferase, followed by the deamination of the 6-amino group of the adenine moiety by a 1-methyladenosine-57 deaminase. This enzymatic pathway differs from that leading to the formation of m1l-37 in the anticodon loop of eukaryotic tRNA(Ala). In the latter case, inosine-37 formation preceeds the S-adenosylmethionine-dependent methylation of l-37 into m1l-37. Thus, enzymatic strategies for catalyzing the formation of 1-methylinosine in tRNAs differ in organisms from distinct evolutionary kingdoms.

Substrate tRNA Recognition Mechanism of Eubacterial tRNA (m1A58) Methyltransferase (TrmI)

“…Our result in the current study reinforces their hypothetical catalytic mechanism: the 6-amino group in A58 is absolutely required for the methyl transfer reaction. In the archaeal modification system, aTrmI methylates both A57 and A58 (21), and the resultant m 1 A57 is further modified to m 1 I57 by deamination (57). Unmethylated I57 has not been found thus far in archaeal tRNAs.…”

Identification of BHB splicing motifs in intron-containing tRNAs from 18 archaea: evolutionary implications

“…It could be that 2Ј-O-methylation of C-34 in anticodon of tRNATrp as well as at many other riboses of the tRNA molecules occurs via an intron-dependent methylation machinery in some archaea (for review, see Omer et al 2003) whereas it might be catalyzed by intron-independent tRNA modifying enzymes in other archaea (Grosjean et al 1995; for review, see Grosjean et al 1997;Constantinesco et al 1999). …”

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

“…Likewise, when a tRNA transcript specific for Phe, Asp, or Ile, lacking modifications, was incubated with AdoMet and cell extracts from P. furiosus or P. abyssi, m 5 U was almost quantitatively formed at position 54 of the TC-loop (Constantinesco et al 1999;Urbonavicius et al 2008). Under the same experimental conditions, U54 in the tRNA Ile transcript was shown to be ''doubly'' modified into m 1 C when incubated with cell extracts from H. volcanii (Grosjean et al 1995). The enzyme responsible for the formation of m 5 U54 in P. abyssi was recently identified as PAB_0719 (aTrmU54) (Urbonavicius et al 2008;Auxilien et al 2011).…”

“…The enzyme responsible for the subsequent N1 methylation of the uracil ring of C54 and the corresponding gene has yet to be identified. However, experiments with whole-cell extract of H. volcanii incubated with 32 Pradiolabeled T7-transcript and AdoMet (SAM) showed enzymatic formation of m 1 C54, indicating that the enzyme of interest was SAM dependent (Grosjean et al 1995).…”