Modifications in Thermus thermophilus 23 S ribosomal RNA are centered in regions of RNA-RNA contact.

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.

Identifying the methyltransferases for m(5)U747 and m(5)U1939 in 23S rRNA using MALDI mass spectrometry.

There are three sites of m(5)U modification in Escherichia coli stable RNAs: one at the invariant tRNA position U54 and two in 23S rRNA at the phylogenetically conserved positions U747 and U1939. Each of these sites is modified by its own methyltransferase, and the tRNA methyltransferase, TrmA, is well-characterised. Two open reading frames, YbjF and YgcA, are approximately 30% identical to TrmA, and here we determine the functions of these candidate methyltransferases using MALDI mass spectrometry. A purified recombinant version of YgcA retains its activity and specificity, and methylates U1939 in an RNA transcript in vitro. We were unable to generate a recombinant version of YbjF that retained in vitro activity, so the function of this enzyme was defined in vivo by engineering a ybjF knockout strain. Comparison of the methylation patterns in 23S rRNAs from YbjF(+) and YbjF(-) strains showed that the latter differed only in the lack of the m(5)U747 modification. With this report, the functions of all the E.coli m(5)U RNA methyltransferases are identified, and a more appropriate designation for YbjF would be RumB (RNA uridine methyltransferases B), in line with the recent nomenclature change for YgcA (now RumA).

Detection of pseudouridine and other modifications in tRNA by cyanoethylation and MALDI mass spectrometry.

Mass spectrometry plays a central role in the characterisation of modified nucleotides, but pseudouridine is a mass-silent post-transcriptional modification and hence not detectable by direct mass spectrometric analysis. We show by the use of matrix-assisted laser desorption/ionisation (MALDI) mass spectrometry that pseudouridines in tRNA can be specifically cyanoethylated by acrylonitrile without affecting the uridines. The tRNA was cyanoethylated and then subjected to digestion with either RNase A or RNase T1. Cyanoethylated digestion fragments were identified by mass spectrometric comparison of untreated and acrylonitrile-treated samples, where the addition of one acrylonitrile resulted in a mass increment of 53.0 Da. The exact modified nucleotide could be identified by tandem mass spectrometry on the cyanoethylated digestion fragment. The methodology was used to identify additional one 4-thiouridine and one pseudouridine in tRNA(TyrII) from Escherichia coli. Furthermore, we observed that RNase A is highly tolerant towards nucleotide modifications, only being inhibited by 2'-O-methylation, whereas RNase T1 cleavage is affected by most nucleotide modifications.