Discovery of the β-barrel–type RNA methyltransferase responsible forN⁶-methylation ofN⁶-threonylcarbamoyladenosine in tRNAs

Abstract

Methylation is a versatile reaction involved in the synthesis and modification of biologically active molecules, including RNAs. N6-methyl-threonylcarbamoyl adenosine (m6t6A) is a post-transcriptional modification found at position 37 of tRNAs from bacteria, insect, plants, and mammals. Here, we report that in Escherichia coli, yaeB (renamed as trmO) encodes a tRNA methyltransferase responsible for the N6-methyl group of m6t6A in tRNAThr specific for ACY codons. TrmO has a unique single-sheeted β-barrel structure and does not belong to any known classes of methyltransferases. Recombinant TrmO employs S-adenosyl-L-methionine (AdoMet) as a methyl donor to methylate t6A to form m6t6A in tRNAThr. Therefore, TrmO/YaeB represents a novel category of AdoMet-dependent methyltransferase (Class VIII). In a ΔtrmO strain, m6t6A was converted to cyclic t6A (ct6A), suggesting that t6A is a common precursor for both m6t6A and ct6A. Furthermore, N6-methylation of t6A enhanced the attenuation activity of the thr operon, suggesting that TrmO ensures efficient decoding of ACY. We also identified a human homolog, TRMO, indicating that m6t6A plays a general role in fine-tuning of decoding in organisms from bacteria to mammals.

Diversity of the biosynthesis pathway for threonylcarbamoyladenosine (t6A), a universal modification of tRNA

“…63 The limited distribution of m 6 t 6 A is confounded by the small number of organisms in which tRNAs have been sequenced. 7 m 6 t 6 A is formed by TsaA (E. coli YaeB was recently identified as the gene responsible for TsaA activity, and renamed TrmO) 64 by transferring a methyl group from S-adenosylmethionine (AdoMet) to tRNA Thr (GGU) containing t 6 A. 63 TrmO has a unique single-sheeted b-barrel structure and does not belong to any known classes of methyltransferases, representing a novel category of AdoMet-dependent methyltransferase (Class VIII).…”

“…64 m 6 t 6 A slightly improves translational efficiency at the codon ACY. 63,64 TrmO is widely distributed throughout life and cross-kingdom functional analysis was performed to show the activity was conserved. 64 ( Fig.…”

“…63,64 TrmO is widely distributed throughout life and cross-kingdom functional analysis was performed to show the activity was conserved. 64 ( Fig. 2) The third known derivative of t 6 A, ms 2 t 6 A, is found only on tRNA Lys (UUU) in a subset of organisms.…”

Nucleoside Analysis by Hydrophilic Interaction Liquid Chromatography Coupled with Mass Spectrometry

“…Reverse-phase chromatography (RPC) coupled with electrospray ionization (ESI) mass spectrometry (MS) has been used to analyze RNA modifications for several decades (Gaston & Limbach, 2014;Pomerantz & McCloskey, 1990;Su et al, 2014;Suzuki, Ikeuchi, Noma, Suzuki, & Sakaguchi, 2007). Our group has employed this technique combined with reverse genetics for the identification of novel genes responsible for the biogenesis of RNA modifications (Ikeuchi, Kitahara, & Suzuki, 2008;Ikeuchi, Shigi, Kato, Nishimura, & Suzuki, 2006;Kimura, Ikeuchi, Kitahara, Sakaguchi, & Suzuki, 2012;Kimura & Suzuki, 2010;Kimura et al, 2014;Noma, Kirino, Ikeuchi, & Suzuki, 2006;Noma, Sakaguchi, & Suzuki, 2009;Soma et al, 2003;Suzuki et al, 2007). However, RPC/ESI-MS is not always suitable for detecting hydrophilic or polar compounds.…”

Structural and functional characterization of the TYW3/Taw3 class of SAM-dependent methyltransferases

“…The majority of methyltransferases use S-adenosyl-L-methionine (SAM) as a methyl donor (Kozbial and Mushegian 2005). SAM-dependent methyltransferases can be divided into eight classes, based on their unique sequence and structural features (Schubert et al 2003;Kozbial and Mushegian 2005;Kaminska et al 2010;Kimura et al 2014). The largest classes contain a Rossman fold (class I) or a SET domain (class V) (Kozbial and Mushegian 2005).…”