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Complete characterization of a biomolecule's chemical structure is crucial in the full understanding of the relations between their structure and function. The dominating components in ribosomes are ribosomal RNAs (rRNAs), and the entire rRNA—but a single modified nucleoside at position 2501 in 23S rRNA—has previously been characterized in the bacterium Escherichia coli. Despite a first report nearly 20 years ago, the chemical nature of the modification at position 2501 has remained elusive, and attempts to isolate it have so far been unsuccessful. We unambiguously identify this last unknown modification as 5-hydroxycytidine—a novel modification in RNA. Identification of 5-hydroxycytidine was completed by liquid chromatography under nonoxidizing conditions using a graphitized carbon stationary phase in combination with ion trap tandem mass spectrometry and by comparing the fragmentation behavior of the natural nucleoside with that of a chemically synthesized ditto. Furthermore, we show that 5-hydroxycytidine is also present in the equivalent position of 23S rRNA from the bacterium Deinococcus radiodurans. Given the unstable nature of 5-hydroxycytidine, this modification might be found in other RNAs when applying the proper analytical conditions as described here.

Methyltransferase enzymes that use S-adenosylmethionine as a cofactor to catalyze 5-methyl uridine (m5U) formation in tRNAs and rRNAs are widespread in Bacteria and Eukaryota, but are restricted to the Thermococcales and Nanoarchaeota groups amongst the Archaea. The RNA m5U methyltransferases appear to have arisen in Bacteria and were then dispersed by horizontal transfer of an rlmD-type gene to the Archaea and Eukaryota. The bacterium Escherichia coli has three gene paralogs and these encode the methyltransferases TrmA that targets m5U54 in tRNAs, RlmC (formerly RumB) that modifies m5U747 in 23S rRNA, and RlmD (formerly RumA) the archetypical enzyme that is specific for m5U1939 in 23S rRNA. The thermococcale archaeon Pyrococcus abyssi possesses two m5U methyltransferase paralogs, PAB0719 and PAB0760, with sequences most closely related to the bacterial RlmD. Surprisingly, however, neither of the two P. abyssi enzymes displays RlmD-like activity in vitro. PAB0719 acts in a TrmA-like manner to catalyze m5U54 methylation in P. abyssi tRNAs, and here we show that PAB0760 possesses RlmC-like activity and specifically methylates the nucleotide equivalent to U747 in P. abyssi 23S rRNA. The findings indicate that PAB0719 and PAB0760 originated as RlmD-type m5U methyltransferases and underwent changes in target specificity after their acquisition by a Thermococcales ancestor from a bacterial source.

Ribosomal RNA from all organisms contains post-transcriptionally modified nucleotides whose function is far from clear. To gain insight into the molecular interactions of modified nucleotides, we investigated the modification status of Thermus thermophilus 5 S and 23 S ribosomal RNA by mass spectrometry and chemical derivatization/primer extension. A total of eleven modified nucleotides was found in 23 S rRNA, of which eight were singly methylated nucleotides and three were pseudouridines. These modified nucleotides were mapped into the published three-dimensional ribosome structure. Seven of the modified nucleotides located to domain IV, and four modified nucleotides located to domain V of the 23 S rRNA. All posttranscriptionally modified nucleotides map in the center of the ribosome, and none of them are in contact with ribosomal proteins. All except one of the modified nucleotides were found in secondary structure elements of the 23 S ribosomal RNA that contact either 16 S ribosomal RNA or transfer RNA, with five of these nucleotides physically involved in intermolecular RNA-RNA bridges. These findings strongly suggest that the post-transcriptional modifications play a role in modulating intermolecular RNA-RNA contacts, which is the first suggestion on a specific function of endogenous ribosomal RNA modifications.

Post-transcriptional modifications were mapped in domains II, IV and V of 23S RNA from the archaeon Haloarcula marismortui. The RNA was investigated by two primer extension techniques using reverse transcriptase and three mass spectrometry techniques. One primer extension technique utilized decreasing concentrations of deoxynucleotide triphosphates to detect 2'-O-ribose methylations and other polymerase blocking modifications. In the other, the rRNA was chemically modified, followed by mild alkaline hydrolysis to map pseudo-uridine groups (Psis). RNA fragments for mass spectrometry were isolated from 23S rRNA by site-directed RNase H or mung bean nuclease digestion followed by gel purification. Modified RNase digestion fragments were identified with matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) and the modifications were further studied by tandem MS. Psis suggested by the primer extension technique were verified by specific cyanoethylation and mass spectrometric detection. A total of only five post-transcriptionally methylated nucleotides and three Psis were detected in the three 23S rRNA domains. One of the methylated nucleotides has not been reported while a dispute about the number of Psis is solved. The limited number of modified nucleotides suggests that H. marismortui does not have special needs for extensive rRNA modifications. We have performed detailed investigations on the three-dimensional location and molecular interactions of the modified nucleotides by computer analysis. Our results show that all the modified positions are at regions with RNA-RNA contacts and all except one are at the surface of the subunit and in functionally important regions.