Discovery and biological characterization of geranylated RNA in bacteria

Abstract

A general mass spectrometry-based screen for unusually hydrophobic cellular small-molecule RNA conjugates revealed geranylated RNA in E. coli, Enterobacter aerogenes, Pseudomonas aeruginosa, and Salmonella thyphimurium. The geranyl group is conjugated to the sulfur atom in two 5-methylaminomethyl-2-thiouridine nucleotides. These geranylated nucleotides occur in the first anticodon position of tRNAGluUUC, tRNALysUUU, tRNAGlnUUG at a frequency of up to 6.7% (~400 geranylated nucleotides per cell). RNA geranylation levels can be increased or abolished by mutation or deletion of the selU (ybbB) gene in E. coli, and purified SelU protein in the presence of geranyl pyrophosphate and tRNA can produce geranylated tRNA. The presence or absence of the geranyl group in tRNAGluUUC, tRNALysUUU, and tRNAGlnUUG affects codon bias and frameshifting during translation. These RNAs represent the first reported examples of oligoisoprenylated cellular nucleic acids.

Prenyltransferases as key enzymes in primary and secondary metabolism

“…Prenyltransferases catalyze the transfer reactions of prenyl moieties from different prenyl donors, e.g., dimethylallyl (DMAPP with a branched C 5 -chain), geranyl (GPP, C 10 ), farnesyl (FPP,C 15 ), or geranylgeranyl (GGPP, C 20 ) diphosphate, to various aliphatic or aromatic acceptors of both low and high molecular substances including proteins and nucleic acids (Dumelin et al 2012;Heide 2009a;Li 2009a;Oldfield and Lin 2012;Palsuledesai and Distefano 2015;Xie et al 2007;Yazaki et al 2009). The prenylation reactions can take place in regular manner (regular prenylation) by connection of the prenyl moieties via their C-1 to an acceptor or reverse manner (reverse prenylation) via their C-3 atoms (Heide 2009a;.…”

Distribution and frequencies of post-transcriptional modifications in tRNAs

“…One big challenge in the analysis of tRNA modifications concerns the "degree" of modification one can expect for the hypermodified residues (understood as the fraction of experimentally characterized tRNA sequences that contain this modified residue). Partial character of certain modified bases has been fully documented in some cases, [41][42][43][44][45] but the information can usually be retrieved only from the original publications describing the sequencing data, and it is rarely quantitative. Partial modification was often pointed out when the tRNA to be sequenced originated from cells cultivated in different physiological conditions (e.g., aerobic vs anaerobic, different temperature of growth, availability of intermediate metabolites or cofactors, stress conditions, tumor malignancy etc.).…”

The identification and characterization of non-coding and coding RNAs and their modified nucleosides by mass spectrometry

“…Besides E. coli, the geranylated modifications has also been shown to be present in other bacteria including Enterobacter aerogenes, Pseudomonas aeruginosa, and Salmonella typhimurium. 18 The discovery of agmatidine, cyanomethyluridine and these geranylated uridines is consistent with the cellular use of metabolic intermediates and products as reagents for RNA modification. 19 A rather unique example of an RNA modification that appears to have been overlooked for many years was reported by Suzuki and colleagues.…”

“…1) have been identified at position 34, the wobble position, where a large geranyl group is found on the sulfur at the second position of the base. 17,18 This modification was found to be catalyzed by selU through MS analysis of nucleosides from knock out strains and in vitro activity. Besides E. coli, the geranylated modifications has also been shown to be present in other bacteria including Enterobacter aerogenes, Pseudomonas aeruginosa, and Salmonella typhimurium.…”

“…79 One advantage of the CARD approach for mapping modifications onto RNAs is that rather complex mixtures, such as mixtures of total tRNAs, can be analyzed without requiring the de novo sequencing of all detected digestion products nor requiring the purification of individual RNAs prior to analysis by techniques such as Chaplet chromatography. 49 However, a limitation of this CARD approach arises when 18 O water is used for isotopic labeling. The small mass difference (C2 Da) between the reference and unknown digestion products become challenging to characterize due to the presence of natural isotopes (e.g., 13 C and 15 N), which can overlap and obscure the doublets expected from identical digestion products.…”