Identification and codon reading properties of 5-cyanomethyl uridine, a new modified nucleoside found in the anticodon wobble position of mutant haloarchaeal isoleucine tRNAs

Abstract

Most archaea and bacteria use a modified C in the anticodon wobble position of isoleucine tRNA to base pair with A but not with G of the mRNA. This allows the tRNA to read the isoleucine codon AUA without also reading the methionine codon AUG. To understand why a modified C, and not U or modified U, is used to base pair with A, we mutated the C34 in the anticodon of Haloarcula marismortui isoleucine tRNA (tRNA 2 Ile ) to U, expressed the mutant tRNA in Haloferax volcanii, and purified and analyzed the tRNA. Ribosome binding experiments show that although the wild-type tRNA 2 Ile binds exclusively to the isoleucine codon AUA, the mutant tRNA binds not only to AUA but also to AUU, another isoleucine codon, and to AUG, a methionine codon. The G34 to U mutant in the anticodon of another H. marismortui isoleucine tRNA species showed similar codon binding properties. Binding of the mutant tRNA to AUG could lead to misreading of the AUG codon and insertion of isoleucine in place of methionine. This result would explain why most archaea and bacteria do not normally use U or a modified U in the anticodon wobble position of isoleucine tRNA for reading the codon AUA. Biochemical and mass spectrometric analyses of the mutant tRNAs have led to the discovery of a new modified nucleoside, 5-cyanomethyl U in the anticodon wobble position of the mutant tRNAs. 5-Cyanomethyl U is present in total tRNAs from euryarchaea but not in crenarchaea, eubacteria, or eukaryotes.

A Platform for Discovery and Quantification of Modified Ribonucleosides in RNA

“…For example, mass spectrometric methods have been used to discover a variety of new tRNA modifications in the past several years, such as 5-cyanomethyl uridine (cnm 5 U), cyclic N 6 -threonylcarbamoyladenosine (ct 6 A), geranylated 2-thiouridine (ges 2 U), geranylated 5-methylaminomethyl-2-thiouridine (mnm 5 ges 2 U), and geranylated 5-carboxymethylaminomethyl-2-thiouridine (cmnm 5 ges 2 U) and N 6 , N 6 -dimethyladenosine (m 6,6 A). Two of these (cnm 5 U and ct 6 A) were discovered by hypothesis-driven, targeted LC-MS/MS of unknown modified tRNA bases from isolated tRNA isoacceptors (Mandal et al, 2014; Miyauchi et al, 2013), while the other four were identified by data-driven chemical screens for ribonucleoside variants (C. T. Chan et al, 2011; Chen, Kowtoniuk, Agarwal, Shen, & Liu, 2009; Kellner, Neumann, et al, 2014; Su et al, 2014).…”

“…Together, integration of datasets from both neutral loss analysis and MFE-MSC screens provides a list of modified ribonucleoside candidates that can be confirmed by a variety of methods, including analysis of commercially available standard compounds, high mass-accuracy tandem MS (Lim, Chen, Sensenhauser, Cook, & Subrahmanyam, 2007), ion trap MS n mass spectrometry, controlled in-source fragmentation in space (pseudo-MS 3 ) (C. T. Chan et al, 2011; Mandal et al, 2014), homonuclear 1 H-2D-NMR (COSY, TOCSY and NOESY) or heteronuclear 1 H- 13 C 2D-NMR (HMQC) (Reynolds & Enriquez, 2002), and by their characteristic chemical derivatization reactions (Kellner, Burhenne, & Helm, 2010). …”

Nucleoside Analysis by Hydrophilic Interaction Liquid Chromatography Coupled with Mass Spectrometry

“…They have a wide variety of chemical diversity including methylation, hydroxylation, acetylation, deamination, isomerization, selenylation, reduction, cyclization, and conjugation with amino acids and sugars (Machnicka et al, 2013). In the past few years, our laboratory and others have reported several novel RNA modifications (Dumelin, Chen, Leconte, Chen, & Liu, 2012;Ikeuchi et al, 2010;Mandal et al, 2014;Miyauchi, Kimura, & Suzuki, 2013). The chemical space of RNA modification will likely expand with the development of improved analytical technologies.…”

Nonsense suppression in archaea

“…When necessary, novobiocin, thymidine, and uracil were added to a concentration of 0.2, 40, or 50 μg/mL, respectively. Transformation of H. volcanii mediated by PEG 600 was as described before (48).…”