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), andSulfolobus 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 mesophilicMethanococcus maripaludisand the most complex one inSulfolobus acidocaldarius. Notable observations are the frequent occurrence of ac⁴C nucleotides in thermophilic archaeal tRNAs, the presence of m⁷G at positions 1 and 10 inPyrococcus furiosustRNAs, 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 fromMethanocaldococcus jannaschiiandHaloferax volcanii.

RNA modifications are involved in numerous biological processes and are present in all RNA classes. These modifications can be constitutive or modulated in response to adaptive processes. RNA modifications play multiple functions since they can impact RNA base-pairings, recognition by proteins, decoding, as well as RNA structure and stability. However, their roles in stress, environmental adaptation and during infections caused by pathogenic bacteria have just started to be appreciated. With the development of modern technologies in mass spectrometry and deep sequencing, recent examples of modifications regulating host-pathogen interactions have been demonstrated. They show how RNA modifications can regulate immune responses, antibiotic resistance, expression of virulence genes, and bacterial persistence. Here, we illustrate some of these findings, and highlight the strategies used to characterize RNA modifications, and their potential for new therapeutic applications.