Systems‐Based Analysis of Modified tRNA Bases

Abstract

The genetic system contains several levels of information. Firstly, the sequence of the canonical bases A, C, G, and T/U in DNA and RNA encodes amino acids through specific base triplets. Secondly, the methylation status of the cytosine base in DNA imprints epigenetic information into the genetic system, thereby contributing to the division of genes into active and inactive elements. Both information layers are chemically well investigated. Less is known about a putative third level of information associated with the chemical modification of RNA nucleobases. Although RNA, and in particular tRNA, is known to contain more than 100 different modified nucleosides, [1] the exact type of information added by base modification is largely unknown. A number of common modifications have been shown to improve the maintenance of the reading frame, [2] influence RNA stability, [3] and to be involved in proof-reading by tRNA synthetases. [4] Recently, it was discovered that the collective set of modified tRNA nucleosides is a regulated component of stress response and gives us a first hint that cells may actively adjust the modification pattern in response to external factors. [5] To learn more about the function of modified nucleobases we have investigated relationships between species by quantification of the modification content. By using a parallel systems-type approach we discovered that the collective set of modified bases is highly species-specific and linked to phylogeny. These data then enabled us to calculate a detailed phylogenetic tree that is consistent with those obtained from traditional data such as the homology of rRNA sequences, [6] conserved orthologous genes, [7] sequences of tRNA synthetases, [8] and tRNA-dependent amidotransferases.[9] The result shows that the set of base modifications is not universal, but rather a highly species-specific code under evolutionary selection to appropriately match base triplets with the corresponding cognate amino acids.For the study, we applied our recently developed LC-MSbased method for the quantification of modified nucleosides by using isotopically labeled standards.[10] For the parallel quantification we synthesized 18 tRNA modifications in both their natural and isotopically labeled forms (Scheme 1). A number of these nucleosides are present in the 3'-position to the anticodon in position 37, while others are distributed through the tRNA structure. The modifications studied are involved in a range of biological processes such as structural stabilization, codon binding, and translation initiation. [2a, 3, 10b, 11] With these tRNA nucleosides in hand, we analyzed the tRNA modification pattern of 16 species, so as to cover several branches of the phylogenetic tree. These species include five eukaryotes as well as five Gram-negative proteobacteria and five Gram-positive bacteria of the firmicutes. In addition we studied the bacterium Deinococcus radiodurans, which has a somewhat ambiguous classification. D. radiodurans is typically identified as Gram-positive, but ...

Evaluating the reproducibility of quantifying modified nucleosides from ribonucleic acids by LC–UV–MS

“…Recently, accurate relative and absolute quantification of modified nucleosides has been demonstrated through the use of stable-isotope labeling [40–42]. Although the use of stable-isotope labeling can yield accurate and precise measurements on the number of modified nucleosides present in the sample, it cannot be used in a general strategy for total nucleoside analysis unless the specific modifications are limited to those with stable-isotope analogs [40,41] or the organism can easily be cultured in labeled media [42]. …”

Structures of Human ALKBH5 Demethylase Reveal a Unique Binding Mode for Specific Single-stranded N6-Methyladenosine RNA Demethylation

“…In higher eukaryotic organisms, more than 100 distinct modifications have been identified in cellular mRNA, tRNA, and rRNA (1,2). Among these modifications, N 6 -methylated adenosine (m 6 A) 3 is the most prevalent internal modification in mRNA; m 6 A has been identified in many eukaryotes and viruses (3,4).…”

Mass spectrometry of modified RNAs: recent developments

“…Carell and co-workers have reported the synthesis of multiple modified ribonucleosides, all of which can incorporate one or more isotopic labels. 36, 37 An alternative strategy was established by Helm and co-workers, wherein standards for modified ribonucleosides are generated by sample culturing in isotopically labeled medium. 38 The added advantage of this latter approach is that the diversity of standards is only limited by the organism’s inherent modification machinery.…”