Influence of Phylogeny on Posttranscriptional Modification of rRNA in Thermophilic Prokaryotes:  The Complete Modification Map of 16S rRNA of Thermus thermophilus

Abstract

Posttranscriptional modification in RNA generally serves to fine-tune and regulate RNA structure and, in many cases, is relatively conserved and phylogenetically distinct. We report the complete modification map for SSU rRNA from Thermus thermophilus, determined primarily by HPLC/electrospray ionization MS-based methods. Thermus modification levels are significantly lower, and structures at the nucleoside level are very different from those of the archaeal thermophile Sulfolobus solfataricus growing in the same temperature range [Noon, K. R., et al. (1998) J. Bacteriol. 180, 2883-2888]. The Thermus modification map is unexpectedly similar to that of Escherichia coli (11 modified sites), with which it shares identity in 8 of the 14 modifications. Unlike the heavily methylated Sulfolobus SSU RNA, Thermus contains a single ribose-methylated residue, N(4),2'-O-dimethylcytidine-1402, suggesting that O-2'-ribose methylation in this bacterial thermophile plays a reduced role in thermostabilization compared with the thermophilic archaea. Adjacent pseudouridine residues were found in the single-stranded 3' tail of Thermus 16S rRNA at residues 1540 and 1541 (E. coli numbering) in the anti-Shine-Dalgarno mRNA binding sequence. The present results provide an example of the potential of LC/MS for extensive modification mapping in large RNAs.

Modification of 16S ribosomal RNA by the KsgA methyltransferase restructures the 30S subunit to optimize ribosome function

“…While an additional methylation at the N2 of G1516 is found in helix 45 of some bacteria but is absent from T. thermophilus (Guymon et al 2006), it does not alter loop thermal stability or interfere with hydrogen bonding ).…”

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

“…[38][39][40] RNA modification mapping for an individual RNA typical begins by obtaining information on all of the modified nucleosides present in the RNA through LC-MS/MS analysis of nucleoside digests (represented by the left side of Fig. 5), which was described above.…”

“…The number of characterized bovine mitochondrial tRNAs was increased from 11 to 22 tRNAs, and 5 tRNAs that were previously characterized were found to contain www.landesbioscience.comadditional modifications that were originally not reported or reported as unknown modifications. 50 McCloskey and coworkers, building extensively on their RNA modification mapping protocol, 37 have mapped modified nucleosides onto the sequences of 16S rRNA from Haloferax volcanii, 51 Thermus thermophilus, 39 and Thermotoga maritima 40 by LC-MS/MS. Also using LC-MS/MS, Rozenski and coworkers mapped modifications onto the 16S rRNA sequences of Clostridium acetobutylicum 52 and Legionella pneumophila.…”

Detection of RNA modifications

“…Presence of pseudouridine causes the signals m/z 225, 207, 189, 165 or 164 to appear in the mass spectra. 37 A particularly sophisticated recent approach identified RNA conjugates of CoA and NAD by a combination of chemical treatment with alkali and nucelophiles and comparative high-resolution liquid chromatography and mass spectrometry. 38,39 Yet another recently evolved method is nucleoside-specific derivatization (e.g., of pseudouridine with acrylonitrile, with N-cyclohexyl-N'-β-(4-methylmorpholinium) ethylcarbodiimide p-tosylate (CMCT), or with methylvinylsulfone, see also Table 1) followed by MS analysis.…”