The RNA Modification Database, RNAMDB: 2011 update.

Since its inception in 1994, The RNA Modification Database (RNAMDB, http://rna-mdb.cas.albany.edu/RNAmods/) has served as a focal point for information pertaining to naturally occurring RNA modifications. In its current state, the database employs an easy-to-use, searchable interface for obtaining detailed data on the 109 currently known RNA modifications. Each entry provides the chemical structure, common name and symbol, elemental composition and mass, CA registry numbers and index name, phylogenetic source, type of RNA species in which it is found, and references to the first reported structure determination and synthesis. Though newly transferred in its entirety to The RNA Institute, the RNAMDB continues to grow with two notable additions, agmatidine and 8-methyladenosine, appended in the last year. The RNA Modification Database is staying up-to-date with significant improvements being prepared for inclusion within the next year and the following year. The expanded future role of The RNA Modification Database will be to serve as a primary information portal for researchers across the entire spectrum of RNA-related research.

Isolation and posttranscriptional modification analysis of native BC1 RNA from mouse brain.

We present a simple and general affinity method, based on size fractionation and nucleic acid complementarity, to isolate sufficient amounts of native RNA molecules for further physicochemical studies, such as modification state of nucleotides. In the case presented here, we purified four micrograms of dendritic neuronal BC1 RNA from 130 grams of mouse brain (initially yielding a total of 200 mg RNA). Directly combined liquid chromatography-electrospray ionization mass spectrometry (LC/MS) analysis revealed no base or sugar backbone modifications in native BC1 RNA, despite earlier indications that C-54 could be methylated in vitro (Cm5, position 54).

Post-transcriptional modifications in the small subunit ribosomal RNA from Thermotoga maritima, including presence of a novel modified cytidine.

Post-transcriptional modifications of RNA are nearly ubiquitous in the principal RNAs involved in translation. However, in the case of rRNA the functional roles of modification are far less established than for tRNA, and are subject to less knowledge in terms of specific nucleoside identities and their sequence locations. Post-transcriptional modifications have been studied in the SSU rRNA from Thermotoga maritima (optimal growth 80 degrees C), one of the most deeply branched organisms in the Eubacterial phylogenetic tree. A total of 10 different modified nucleosides were found, the greatest number reported for bacterial SSU rRNA, occupying a net of approximately 14 sequence sites, compared with a similar number of sites recently reported for Thermus thermophilus and 11 for Escherichia coli. The relatively large number of modifications in Thermotoga offers modest support for the notion that thermophile rRNAs are more extensively modified than those from mesophiles. Seven of the Thermotoga modified sites are identical (location and identity) to those in E. coli. An unusual derivative of cytidine was found, designated N-330 (Mr 330.117), and was sequenced to position 1404 in the decoding region of the rRNA. It was unexpectedly found to be identical to an earlier reported nucleoside of unknown structure at the same location in the SSU RNA of the archaeal mesophile Haloferax volcanii.

Influence of phylogeny on posttranscriptional modification of rRNA in thermophilic prokaryotes: the complete modification map of 16S rRNA of Thermus thermophilus.

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.

N6-Acetyladenosine: a new modified nucleoside from Methanopyrus kandleri tRNA.

Post-transcriptionally modified nucleosides are constituents of transfer RNA (tRNA) that are known to influence tertiary structure, stability and coding properties. Modifications in unfractionated tRNA from the phylogenetically unique archaeal methanogen Methanopyrus kandleri (optimal growth temperature 98 degrees C) were studied using liquid chromatography-mass spectrometry to establish the extent to which they might differ from those of other methanogens. The exceptionally diverse population of nucleosides included four new nucleosides of unknown structure, and one that was characterized as N(6)-acetyladenosine, a new RNA constituent. The nucleoside modification pattern in M. kandleri tRNA is notably different from that of other archaeal methanogens, and is closer to that of the thermophilic crenarchaeota.

A "gain of function" mutation in a protein mediates production of novel modified nucleosides.

The mutation sufY204 mediates suppression of a +1 frameshift mutation in the histidine operon of Salmonella enterica serovar Typhimurium and synthesis of two novel modified nucleosides in tRNA. The sufY204 mutation, which results in an amino-acid substitution in a protein, is, surprisingly, dominant over its wild-type allele and thus it is a "gain of function" mutation. One of the new nucleosides is 5-methylaminomethyl-2-thiouridine (mnm(5)s(2)U34) modified by addition of a C(10)H(17) side chain of unknown structure. Increased amounts of both nucleosides in tRNA are correlated to gene dosage of the sufY204 allele, to an increased efficiency of frameshift suppression, and to a decreased amount of the wobble nucleoside mnm(5)s(2)U34 in tRNA. Purified tRNA(Gln)(cmnm(5)s(2)UUG) in the mutant strain contains a modified nucleoside similar to the novel nucleosides and the level of aminoacylation of tRNA(Gln)(cmnm(5)s(2)UUG) was reduced to 26% compared to that found in the wild type (86%). The results are discussed in relation to the mechanism of reading frame maintenance and the evolution of modified nucleosides in tRNA.

Number, position, and significance of the pseudouridines in the large subunit ribosomal RNA of Haloarcula marismortui and Deinococcus radiodurans.

The number and position of the pseudouridines of Haloarcula marismortui and Deinococcus radiodurans large subunit RNA have been determined by a combination of total nucleoside analysis by HPLC-mass spectrometry and pseudouridine sequencing by the reverse transcriptase method and by LC/MS/MS. Three pseudouridines were found in H. marismortui, located at positions 1956, 1958, and 2621 corresponding to Escherichia coli positions 1915, 1917, and 2586, respectively. The three pseudouridines are all in locations found in other organisms. Previous reports of a larger number of pseudouridines in this organism were incorrect. Three pseudouridines and one 3-methyl pseudouridine (m3Psi) were found in D. radiodurans 23S RNA at positions 1894, 1898 (m3Psi), 1900, and 2584, the m3Psi site being determined by a novel application of mass spectrometry. These positions correspond to E. coli positions 1911, 1915, 1917, and 2605, which are also pseudouridines in E. coli (1915 is m3Psi). The pseudouridines in the helix 69 loop, residues 1911, 1915, and 1917, are in positions highly conserved among all phyla. Pseudouridine 2584 in D. radiodurans is conserved in eubacteria and a chloroplast but is not found in archaea or eukaryotes, whereas pseudouridine 2621 in H. marismortui is more conserved in eukaryotes and is not found in eubacteria. All the pseudoridines are near, but not exactly at, nucleotides directly involved in various aspects of ribosome function. In addition, two D. radiodurans Psi synthases responsible for the four Psi were identified.

An aminoacyl-tRNA synthetase that specifically activates pyrrolysine.

Pyrrolysine, the 22nd cotranslationally inserted amino acid, was found in the Methanosarcina barkeri monomethylamine methyltransferase protein in a position that is encoded by an in-frame UAG stop codon in the mRNA. M. barkeri encodes a special amber suppressor tRNA (tRNA(Pyl)) that presumably recognizes this UAG codon. It was reported that Lys-tRNA(Pyl) can be formed by the aminoacyl-tRNA synthetase-like M. barkeri protein PylS [Srinivasan, G., James, C. M. & Krzycki, J. A. (2002) Science 296, 1459-1462], whereas a later article showed that Lys-tRNA(Pyl) is synthesized by the combined action of LysRS1 and LysRS2, the two different M. barkeri lysyl-tRNA synthetases. Pyrrolysyl-tRNA(Pyl) formation was presumed to result from subsequent modification of lysine attached to tRNA(Pyl). To investigate whether pyrrolysine can be directly attached to tRNA(Pyl) we chemically synthesized pyrrolysine. We show that PylS is a specialized aminoacyl-tRNA synthetase for charging pyrrolysine to tRNA(Pyl); lysine and tRNA(Lys) are not substrates of the enzyme. In view of the properties of PylS we propose to name this enzyme pyrrolysyl-tRNA synthetase. In contrast, the LysRS1:LysRS2 complex does not recognize pyrrolysine and charges tRNA(Pyl) with lysine. These in vitro data suggest that Methanosarcina cells have two pathways for acylating the suppressor tRNA(Pyl). This would ensure efficient translation of the in-frame UAG codon in case of pyrrolysine deficiency and safeguard the biosynthesis of the proteins whose genes contain this special codon.

A truncated aminoacyl-tRNA synthetase modifies RNA.

Aminoacyl-tRNA synthetases are modular enzymes composed of a central active site domain to which additional functional domains were appended in the course of evolution. Analysis of bacterial genome sequences revealed the presence of many shorter aminoacyl-tRNA synthetase paralogs. Here we report the characterization of a well conserved glutamyl-tRNA synthetase (GluRS) paralog (YadB in Escherichia coli) that is present in the genomes of >40 species of proteobacteria, cyanobacteria, and actinobacteria. The E. coli yadB gene encodes a truncated GluRS that lacks the C-terminal third of the protein and, consequently, the anticodon binding domain. Generation of a yadB disruption showed the gene to be dispensable for E. coli growth in rich and minimal media. Unlike GluRS, the YadB protein was able to activate glutamate in presence of ATP in a tRNA-independent fashion and to transfer glutamate onto tRNA(Asp). Neither tRNA(Glu) nor tRNA(Gln) were substrates. In contrast to canonical aminoacyl-tRNA, glutamate was not esterified to the 3'-terminal adenosine of tRNA(Asp). Instead, it was attached to the 2-amino-5-(4,5-dihydroxy-2-cyclopenten-1-yl) moiety of queuosine, the modified nucleoside occupying the first anticodon position of tRNA(Asp). Glutamyl-queuosine, like canonical Glu-tRNA, was hydrolyzed by mild alkaline treatment. Analysis of tRNA isolated under acidic conditions showed that this novel modification is present in normal E. coli tRNA; presumably it previously escaped detection as the standard conditions of tRNA isolation include an alkaline deacylation step that also causes hydrolysis of glutamyl-queuosine. Thus, this aminoacyl-tRNA synthetase fragment contributes to standard nucleotide modification of tRNA.

Tandem mass spectrometry for structure assignments of wye nucleosides from transfer RNA.

The tricyclic wye nucleoside family of eight known members constitutes one of the most complex and interesting series of posttranscriptionally modified nucleosides in transfer RNA. The principal reaction paths represented in collision-induced dissociation mass spectra of wye bases and their analogs have been studied in order to determine those structural features that can be readily established by mass spectrometry. The main routes of fragmentation are determined by the presence vs. absence of an amino acid side chain at C-7 (1H-imidazo[1,2-a]purine nomenclature). The common methionine-related side chain is cleaved at two points, providing a ready means of establishing the presence and net level of side chain modification. For those molecules without a side chain, the initial reaction steps are characteristically controlled by the presence vs. absence of methyl at N-4, allowing determination of the methylation status of that site. In the latter case initial opening of the central (pyrimidine) ring, in analogy to the dissociation behavior of guanine, causes loss of identity of C-6/C-7 so that placement of a single methyl at either site is not possible. Subsequent complex reaction paths follow, which include loss of CO and sequential loss of two molecules of HCN.

The tylosin-resistance methyltransferase RlmA(II) (TlrB) modifies the N-1 position of 23S rRNA nucleotide G748.

The methyltransferase RlmA(II) (TlrB) confers resistance to the macrolide antibiotic tylosin in the drug-producing strain Streptomyces fradiae. The resistance conferred by RlmA(II) is highly specific for tylosin, and no resistance is conferred to other macrolide drugs, or to lincosamide and streptogramin B (MLS(B)) drugs that bind to the same region on the bacterial ribosome. In this study, the methylation site of RlmA(II) is identified unambiguously by liquid chromatography/electrospray ionization mass spectrometry as the N-1 position of 23S rRNA nucleotide G748. This position is contacted by the mycinose sugar moiety of tylosin, which is absent from the other drugs. The selective resistance to tylosin conferred by m(1)G748 illustrates how differences in drug structure facilitate the drug fit at the MLS(B)-binding site. This observation is of relevance for the rational design of novel antimicrobials targeting the MLS(B) site, especially if the antimicrobials are to be used against pathogens possessing m(1)G748.

A novel method for sequence placement of modified nucleotides in mixtures of transfer RNA.

Sequence placement of post-transcriptionally modified nucleosides in tRNA can be experimentally difficult, particularly in cases involving new or unexpected modifications or sequence sites. We describe a mass spectrometry-based approach to this problem, involving the following steps: crude isolations of one or several tRNAs by HPLC from an unfractionated tRNA mixture; digestion to oligonucleotide mixtures by RNase T1; analysis by combined HPLC/electrospray ionization-MS for recognition of modifications; and direct gas-phase sequencing of selected targets in the mixture by LC/MS/MS. Isoacceptor identity can be established in favorable cases when tRNA gene sequences are available.

Influence of temperature on tRNA modification in archaea: Methanococcoides burtonii (optimum growth temperature [Topt], 23 degrees C) and Stetteria hydrogenophila (Topt, 95 degrees C).

We report the first study of tRNA modification in psychrotolerant archaea, specifically in the archaeon Methanococcoides burtonii grown at 4 and 23 degrees C. For comparison, unfractionated tRNA from the archaeal hyperthermophile Stetteria hydrogenophila cultured at 93 degrees C was examined. Analysis of modified nucleosides using liquid chromatography-electrospray ionization mass spectrometry revealed striking differences in levels and identities of tRNA modifications between the two organisms. Although the modification levels in M. burtonii tRNA are the lowest in any organism of which we are aware, it contains more than one residue per tRNA molecule of dihydrouridine, a molecule associated with maintenance of polynucleotide flexibility at low temperatures. No differences in either identities or levels of modifications, including dihydrouridine, as a function of culture temperature were observed, in contrast to selected tRNA modifications previously reported for archaeal hyperthermophiles. By contrast, S. hydrogenophila tRNA was found to contain a remarkable structural diversity of 31 modified nucleosides, including nine methylated guanosines, with eight different nucleoside species methylated at O-2' of ribose, known to be an effective stabilizing motif in RNA. These results show that some aspects of tRNA modification in archaea are strongly associated with environmental temperature and support the thesis that posttranscriptional modification is a universal natural mechanism for control of RNA molecular structure that operates across a wide temperature range in archaea as well as bacteria.

Modification of the universally unmodified uridine-33 in a mitochondria-imported edited tRNA and the role of the anticodon arm structure on editing efficiency.

Editing of tRNA has a wide phylogenetic distribution among eukaryotes and in some cases serves to expand the decoding capacity of the target tRNA. We previously described C-to-U editing of the wobble position of the imported tRNA(Trp) in Leishmania mitochondria, which is essential for decoding UGA codons as tryptophan. Here we show the complete set of nucleotide modifications in the anticodon arm of the mitochondrial and cytosolic tRNA(Trp) as determined by electrospray ionization mass spectrometry. This analysis revealed extensive mitochondria-specific posttranscriptional modifications, including the first example of thiolation of U33, the "universally unmodified" uridine. In light of the known rigidity imparted on sugar conformation by thiolation, our discovery of a thiolated U33 suggests that conformational flexibility is not a universal feature of the anticodon structural signature. In addition, the in vivo analysis of tRNA(Trp) variants presented shows a single base-pair reversal in the anticodon stem of tRNA(Trp) is sufficient to abrogate editing in vivo, indicating that subtle changes in anticodon structure can have drastic effects on editing efficiency.