Comparative patterns of modified nucleotides in individual tRNA species from a mesophilic and two thermophilic archaea

Abstract

To improve and complete our knowledge of archaeal tRNA modification patterns, we have identified and compared the modification pattern (type and location) in tRNAs of three very different archaeal species, Methanococcus maripaludis (a mesophilic methanogen), Pyrococcus furiosus (a hyperthermophile thermococcale), and Sulfolobus acidocaldarius (an acidophilic thermophilic sulfolobale). Most abundant isoacceptor tRNAs (79 in total) for each of the 20 amino acids were isolated by two-dimensional gel electrophoresis followed by in-gel RNase digestions. The resulting oligonucleotide fragments were separated by nanoLC and their nucleotide content analyzed by mass spectrometry (MS/MS). Analysis of total modified nucleosides obtained from complete digestion of bulk tRNAs was also performed. Distinct base- and/or ribose-methylations, cytidine acetylations, and thiolated pyrimidines were identified, some at new positions in tRNAs. Novel, some tentatively identified, modifications were also found. The least diversified modification landscape is observed in the mesophilic Methanococcus maripaludis and the most complex one in Sulfolobus acidocaldarius. Notable observations are the frequent occurrence of ac4C nucleotides in thermophilic archaeal tRNAs, the presence of m7G at positions 1 and 10 in Pyrococcus furiosus tRNAs, and the use of wyosine derivatives at position 37 of tRNAs, especially those decoding U1- and C1-starting codons. These results complete those already obtained by others with sets of archaeal tRNAs from Methanocaldococcus jannaschii and Haloferax volcanii.

Post-Transcriptional Modifications of Conserved Nucleotides in the T-Loop of tRNA: A Tale of Functional Convergent Evolution

“…Notice, the presence of m 1 A58 and/or m 1 I57 depends much on the archaeon considered and the type of tRNA analyzed (especially the nature of nucleotide-59, see below). For example, most tRNAs from halophilic Archaea such as Haloferax volcanii display m 1 I57 and unmodified A58 [ 27 , 28 ], while tRNAs of thermophilic and hyperthermophilic Archaea often display m 1 A58 and/or m 1 I57 [ 29 ] and references therein.…”

“…In Archaea , while Gm18 has been experimentally identified in tRNA of different thermophilic organisms [ 29 ], no stand-alone homolog of TrmH/TRM3 has yet been identified. Instead, an alternative pathway involving a C/D box guide has been suggested, as in the case of 2′- O -ribose methylation of C56 of the T-loop (see below) and also for other nucleotides in the anticodon loop and position 50 of the T-arm [ 40 , 41 ].…”

“…Finally, in the case of the hyperthermophilic crenarchaeon Sulfolobus acidocaldarius , s 2 U has been found at position 54 of several tRNAs. In the particular case of tRNA Val , a methylthio U-derivative was also found, but its chemical structure remains elusive [ 29 ]. Examination of the S. acidocaldarius genome reveals the presence of genes coding for TtuA and TtuB responsible for thiolation of U54 but no gene coding for TrmA (catalyzing m 5 U54), Pus10 (catalyzing Ψ54 in addition of Ψ55), and TrmY (catalyzing m 1 Ψ54) was found.…”

Conservation of Archaeal C/D Box sRNA-Guided RNA Modifications

“…Interestingly, a tRNA-targeting sRNA can mediate methylation of either a single or many tRNAs, depending on whether its guide targets a unique sequence like the region surrounding the wobble base in the anticodon (position 34) or a conserved sequence shared across different tRNA families (Lui et al, 2018). Conversely, a few tRNAs in S. acidocaldarius and P. furiosus contain several methylated or predicted methylated nucleotides which have not yet been linked to corresponding sRNAs or stand-alone specific methyltransferases (Wolff et al, 2020). Several computational studies also identified numerous "orphan guides, " which are defined as snoRNA guide sequences that do not show complementary to any known RNA target (Hüttenhofer et al, 2001;Yang et al, 2006;Lui et al, 2018).…”

Changes of the tRNA Modification Pattern during the Development of Dictyostelium discoideum

“…The distribution of nucleotide mismatch and RTa, respectively, can be used not only to identify modified sites but also to predict the identity of the modification; such a mismatch-based method is implemented in HAMR [11,15]. While this approach cannot provide unambiguous proof for the identity of a modification, it can be used as evidence in particular in comparative settings, e.g., when the identity of the modification at a homologous site in another species has been validated e.g., by mass spectrometry [34,35]. While approximately complete profiles of tRNA modifications are not available for most organisms, they are known for a limited number of eukaryotes including yeast, human, and rat [3].…”