The hyperthermophilic partners Nanoarchaeum and Ignicoccus stabilize their tRNA T-loops via different but structurally equivalent modifications.

The universal L-shaped tertiary structure of tRNAs is maintained with the help of nucleotide modifications within the D- and T-loops, and these modifications are most extensive within hyperthermophilic species. The obligate-commensal Nanoarchaeum equitans and its phylogenetically-distinct host Ignicoccus hospitalis grow physically coupled under identical hyperthermic conditions. We report here two fundamentally different routes by which these archaea modify the key conserved nucleotide U54 within their tRNA T-loops. In N. equitans, this nucleotide is methylated by the S-adenosylmethionine-dependent enzyme NEQ053 to form m5U54, and a recombinant version of this enzyme maintains specificity for U54 in Escherichia coli. In N. equitans, m5U54 is subsequently thiolated to form m5s2U54. In contrast, I. hospitalis isomerizes U54 to pseudouridine prior to methylating its N1-position and thiolating the O4-position of the nucleobase to form the previously uncharacterized nucleotide m1s4Ψ. The methyl and thiol groups in m1s4Ψ and m5s2U are presented within the T-loop in a spatially identical manner that stabilizes the 3'-endo-anti conformation of nucleotide-54, facilitating stacking onto adjacent nucleotides and reverse-Hoogsteen pairing with nucleotide m1A58. Thus, two distinct structurally-equivalent solutions have evolved independently and convergently to maintain the tertiary fold of tRNAs under extreme hyperthermic conditions.

The human methyltransferase ZCCHC4 catalyses N6-methyladenosine modification of 28S ribosomal RNA.

RNA methylations are essential both for RNA structure and function, and are introduced by a number of distinct methyltransferases (MTases). In recent years, N6-methyladenosine (m6A) modification of eukaryotic mRNA has been subject to intense studies, and it has been demonstrated that m6A is a reversible modification that regulates several aspects of mRNA function. However, m6A is also found in other RNAs, such as mammalian 18S and 28S ribosomal RNAs (rRNAs), but the responsible MTases have remained elusive. 28S rRNA carries a single m6A modification, found at position A4220 (alternatively referred to as A4190) within a stem-loop structure, and here we show that the MTase ZCCHC4 is the enzyme responsible for introducing this modification. Accordingly, we found that ZCCHC4 localises to nucleoli, the site of ribosome assembly, and that proteins involved in RNA metabolism are overrepresented in the ZCCHC4 interactome. Interestingly, the absence of m6A4220 perturbs codon-specific translation dynamics and shifts gene expression at the translational level. In summary, we establish ZCCHC4 as the enzyme responsible for m6A modification of human 28S rRNA, and demonstrate its functional significance in mRNA translation.

Spatial variability of prokaryotic and viral abundances in the Kermadec and Atacama Trench regions.

Hadal trenches represent the deepest part of the ocean and are dynamic depocenters with intensified prokaryotic activity. Here, we explored the distribution and drivers of prokaryotic and viral abundance from the ocean surface and 40 cm into sediments in two hadal trench regions with contrasting surface productivity. In the water column, prokaryotic and viral abundance decreased with water depth before reaching a rather stable level at ~ 4000 m depth at both trench systems, while virus to prokaryote ratios were increasing with depth, presumably reflecting the declining availability of organic material. Prokaryotic and viral abundances in sediments were lower at the adjacent abyssal sites than at the hadal sites and declined exponentially with sediment depth, closely tracking the attenuation of total organic carbon (TOC) content. In contrast, hadal sediment exhibited erratic depth profiles of prokaryotes and viruses with many subsurface peaks. The prokaryotic abundance correlated well to extensive fluctuations in TOC content at centimeter scale, which were likely caused by recurring mass wasting events. Yet while prokaryotic and viral abundances cross correlated well in the abyssal sediments, there was no clear correlation in the hadal sites. The results suggested that dynamic depositional conditions and higher substrate availability result in a high spatial heterogeneity in viral and prokaryotic abundances in hadal sediments in comparison to more stable abyssal settings. We argue that these conditions enhance the relatively importance of viruses for prokaryotic mortality and carbon recycling in hadal settings.

Detection of internal N7-methylguanosine (m7G) RNA modifications by mutational profiling sequencing.

Methylation of guanosine on position N7 (m7G) on internal RNA positions has been found in all domains of life and have been implicated in human disease. Here, we present m7G Mutational Profiling sequencing (m7G-MaP-seq), which allows high throughput detection of m7G modifications at nucleotide resolution. In our method, m7G modified positions are converted to abasic sites by reduction with sodium borohydride, directly recorded as cDNA mutations through reverse transcription and sequenced. We detect positions with increased mutation rates in the reduced and control samples taking the possibility of sequencing/alignment error into account and use replicates to calculate statistical significance based on log likelihood ratio tests. We show that m7G-MaP-seq efficiently detects known m7G modifications in rRNA with mutational rates up to 25% and we map a previously uncharacterised evolutionarily conserved rRNA modification at position 1581 in Arabidopsis thaliana SSU rRNA. Furthermore, we identify m7G modifications in budding yeast, human and arabidopsis tRNAs and demonstrate that m7G modification occurs before tRNA splicing. We do not find any evidence for internal m7G modifications being present in other small RNA, such as miRNA, snoRNA and sRNA, including human Let-7e. Likewise, high sequencing depth m7G-MaP-seq analysis of mRNA from E. coli or yeast cells did not identify any internal m7G modifications.

Excision of uracil from DNA by hSMUG1 includes strand incision and processing.

Uracil arises in DNA by hydrolytic deamination of cytosine (C) and by erroneous incorporation of deoxyuridine monophosphate opposite adenine, where the former event is devastating by generation of C → thymine transitions. The base excision repair (BER) pathway replaces uracil by the correct base. In human cells two uracil-DNA glycosylases (UDGs) initiate BER by excising uracil from DNA; one is hSMUG1 (human single-strand-selective mono-functional UDG). We report that repair initiation by hSMUG1 involves strand incision at the uracil site resulting in a 3'-α,ß-unsaturated aldehyde designated uracil-DNA incision product (UIP), and a 5'-phosphate. UIP is removed from the 3'-end by human apurinic/apyrimidinic (AP) endonuclease 1 preparing for single-nucleotide insertion. hSMUG1 also incises DNA or processes UIP to a 3'-phosphate designated uracil-DNA processing product (UPP). UIP and UPP were indirectly identified and quantified by polyacrylamide gel electrophoresis and chemically characterised by matrix-assisted laser desorption/ionisation time-of-flight mass-spectrometric analysis of DNA from enzyme reactions using 18O- or 16O-water. The formation of UIP accords with an elimination (E2) reaction where deprotonation of C2' occurs via the formation of a C1' enolate intermediate. A three-phase kinetic model explains rapid uracil excision in phase 1, slow unspecific enzyme adsorption/desorption to DNA in phase 2 and enzyme-dependent AP site incision in phase 3.

Proteomic Changes of Klebsiella pneumoniae in Response to Colistin Treatment and crrB Mutation-Mediated Colistin Resistance.

Polymyxins are increasingly used as the critical last-resort therapeutic options for multidrug-resistant Gram-negative bacteria. Unfortunately, polymyxin resistance has increased gradually over the past few years. Although studies on polymyxin mechanisms are expanding, systemwide analyses of the underlying mechanism for polymyxin resistance and stress response are still lacking. To understand how Klebsiella pneumoniae adapts to colistin (polymyxin E) pressure, we carried out proteomic analysis of a K. pneumoniae strain cultured with different concentrations of colistin. Our results showed that the proteomic responses to colistin treatment in K. pneumoniae involve several pathways, including (i) gluconeogenesis and the tricarboxylic acid (TCA) cycle, (ii) arginine biosynthesis, (iii) porphyrin and chlorophyll metabolism, and (iv) enterobactin biosynthesis. Interestingly, decreased abundances of class A ß-lactamases, including TEM, SHV-11, and SHV-4, were observed in cells treated with colistin. Moreover, we present comprehensive proteome atlases of paired polymyxin-susceptible and -resistant K. pneumoniae strains. The polymyxin-resistant strain Ci, a mutant of K. pneumoniae ATCC BAA 2146, showed a missense mutation in crrB This crrB mutant, which displayed lipid A modification with 4-amino-4-deoxy-l-arabinose (l-Ara4N) and palmitoylation, showed striking increases in the expression of CrrAB, PmrAB, PhoPQ, ArnBCADT, and PagP. We hypothesize that crrB mutations induce elevated expression of the arnBCADTEF operon and pagP via PmrAB and PhoPQ. Moreover, the multidrug efflux pump KexD, which was induced by crrB mutation, also contributed to colistin resistance. Overall, our results demonstrated proteomic responses to colistin treatment and the mechanism of CrrB-mediated colistin resistance, which may offer valuable information on the management of polymyxin resistance.

Mapping of ribosomal 23S ribosomal RNA modifications in Clostridium sporogenes.

All organisms contain RNA modifications in their ribosomal RNA (rRNA), but the importance, positions and exact function of these are still not fully elucidated. Various functions such as stabilizing structures, controlling ribosome assembly and facilitating interactions have been suggested and in some cases substantiated. Bacterial rRNA contains much fewer modifications than eukaryotic rRNA. The rRNA modification patterns in bacteria differ from each other, but too few organisms have been mapped to draw general conclusions. This study maps 23S ribosomal RNA modifications in Clostridium sporogenes that can be characterized as a non-toxin producing Clostridium botulinum. Clostridia are able to sporulate and thereby survive harsh conditions, and are in general considered to be resilient to antibiotics. Selected regions of the 23S rRNA were investigated by mass spectrometry and by primer extension analysis to pinpoint modified sites and the nature of the modifications. Apparently, C. sporogenes 23S rRNA contains few modifications compared to other investigated bacteria. No modifications were identified in domain II and III of 23S rRNA. Three modifications were identified in domain IV, all of which have also been found in other organisms. Two unusual modifications were identified in domain V, methylated dihydrouridine at position U2449 and dihydrouridine at position U2500 (Escherichia coli numbering), in addition to four previously known modified positions. The enzymes responsible for the modifications were searched for in the C. sporogenes genome using BLAST with characterized enzymes as query. The search identified genes potentially coding for RNA modifying enzymes responsible for most of the found modifications.

Identification of the methyltransferase targeting C2499 in Deinococcus radiodurans 23S ribosomal RNA.

The bacterium Deinococcus radiodurans-like all other organisms-introduces nucleotide modifications into its ribosomal RNA. We have previously found that the bacterium contains a Carbon-5 methylation on cytidine 2499 of its 23S ribosomal RNA, which is so far the only modified version of cytidine 2499 reported. Using homology search, we identified the open reading frame DR_0049 as the primary candidate gene for the methyltransferase that modifies cytidine 2499. Mass spectrometric analysis demonstrated that recombinantly expressed DR0049 protein methylates E. coli cytidine 2499 both in vitro and in vivo. We also inactivated the DR_0049 gene in D. radiodurans through insertion of a chloramphenicol resistance cassette. This resulted in complete absence of the cytidine 2499 methylation, which all together demonstrates that DR_0049 encodes the methyltransferase producing m(5)C2499 in D. radiodurans 23S rRNA. Growth experiments disclosed that inactivation of DR_0049 is associated with a severe growth defect, but available ribosome structures show that cytidine 2499 is positioned very similar in D. radiodurans harbouring the modification and E. coli without the modification. Hence there is no obvious structure-based explanation for the requirement for the C2499 posttranscriptional modification in D. radiodurans.

Automated N-glycan profiling of a mutant Trypanosoma rangeli sialidase expressed in Pichia pastoris, using tandem mass spectrometry and bioinformatics.

A mutant Trypanosoma rangeli sialidase, Tr7, expressed in Pichia pastoris, exhibits significant trans-sialidase activity, and has been used for analytical-scale production of sialylated human milk oligosaccharides. Mass spectrometry-based site-specific N-glycoprofiling of Tr7 showed that heterogeneous high-mannose type N-glycans were present at all the five potential N-linked glycosites. N-linked glycans in Tr7 were predominantly neutral oligosaccharides with compositions Man(8-16)GlcNA(c2), but also mono- and di-phosphorylated oligosaccharides in the forms of Man(9-15)P(1)GlcNA(c2) and Man(9-14)P(2)GlcNA(c2), respectively. Some phosphorylated N-linked glycans further contained an additional HexNAc, which has not previously been reported in P. pastoris-expressed proteins. We compiled a method pipeline that combined hydrophilic interaction liquid chromatography enrichment of glycopeptides, high accuracy mass spectrometry and automated interpretation of the mass spectra with in-house developed "MassAI" software, which proved efficient in glycan site microheterogeneity analysis. Functional analysis showed that the deglycosylated Tr7 retained more than 90% of both the sialidase and trans-sialidase activities relative to the glycosylated Tr7.

Recognition of guanosine by dissimilar tRNA methyltransferases.

Guanosines are important for biological activities through their specific functional groups that are recognized for RNA or protein interactions. One example is recognition of N(1) of G37 in tRNA by S-adenosyl-methionine (AdoMet)-dependent tRNA methyltransferases to synthesize m(1)G37-tRNA, which is essential for translational fidelity in all biological domains. Synthesis of m(1)G37-tRNA is catalyzed by TrmD in bacteria and by Trm5 in eukarya and archaea, using unrelated and dissimilar structural folds. This raises the question of how dissimilar proteins recognize the same guanosine. Here we probe the mechanism of discrimination among functional groups of guanosine by TrmD and Trm5. Guanosine analogs were systematically introduced into tRNA through a combination of chemical and enzymatic synthesis. Single turnover kinetic assays and thermodynamic analysis of the effect of each analog on m(1)G37-tRNA synthesis reveal that TrmD and Trm5 discriminate functional groups differently. While both recognize N(1) and O(6) of G37, TrmD places a much stronger emphasis on these functional groups than Trm5. While the exocyclic 2-amino group of G37 is important for TrmD, it is dispensable for Trm5. In addition, while an adjacent G36 is obligatory for TrmD, it is nonessential for Trm5. These results depict a more rigid requirement of guanosine functional groups for TrmD than for Trm5. However, the sensitivity of both enzymes to analog substitutions, together with an experimental revelation of their low cellular concentrations relative to tRNA substrates, suggests a model in which these enzymes rapidly screen tRNA by direct recognition of G37 in order to monitor the global state of m(1)G37-tRNA.

Characterization of an extensin-modifying metalloprotease: N-terminal processing and substrate cleavage pattern of Pectobacterium carotovorum Prt1.

Compared to other plant cell wall-degrading enzymes, proteases are less well understood. In this study, the extracellular metalloprotease Prt1 from Pectobacterium carotovorum (formerly Erwinia carotovora) was expressed in Escherichia coli and characterized with respect to N-terminal processing, thermal stability, substrate targets, and cleavage patterns. Prt1 is an autoprocessing protease with an N-terminal signal pre-peptide and a pro-peptide which has to be removed in order to activate the protease. The sequential cleavage of the N-terminus was confirmed by mass spectrometry (MS) fingerprinting and N-terminus analysis. The optimal reaction conditions for the activity of Prt1 on azocasein were at pH 6.0, 50 °C. At these reaction conditions, K M was 1.81 mg/mL and k cat was 1.82 × 10(7) U M(-1). The enzyme was relatively stable at 50 °C with a half-life of 20 min. Ethylenediaminetetraacetic acid (EDTA) treatment abolished activity; Zn(2+) addition caused regain of the activity, but Zn(2+)addition decreased the thermal stability of the Prt1 enzyme presumably as a result of increased proteolytic autolysis. In addition to casein, the enzyme catalyzed degradation of collagen, potato lectin, and plant extensin. Analysis of the cleavage pattern of different substrates after treatment with Prt1 indicated that the protease had a substrate cleavage preference for proline in substrate residue position P1 followed by a hydrophobic residue in residue position P1' at the cleavage point. The activity of Prt1 against plant cell wall structural proteins suggests that this enzyme might become an important new addition to the toolbox of cell-wall-degrading enzymes for biomass processing.

Identification and characterization of the Thermus thermophilus 5-methylcytidine (m5C) methyltransferase modifying 23 S ribosomal RNA (rRNA) base C1942.

Methylation of cytidines at carbon-5 is a common posttranscriptional RNA modification encountered across all domains of life. Here, we characterize the modifications of C1942 and C1962 in Thermus thermophilus 23 S rRNA as 5-methylcytidines (m(5)C) and identify the two associated methyltransferases. The methyltransferase modifying C1942, named RlmO, has not been characterized previously. RlmO modifies naked 23 S rRNA, but not the assembled 50 S subunit or 70 S ribosomes. The x-ray crystal structure of this enzyme in complex with the S-adenosyl-l-methionine cofactor at 1.7 Å resolution confirms that RlmO is structurally related to other m(5)C rRNA methyltransferases. Key residues in the active site are located similar to the further distant 5-methyluridine methyltransferase RlmD, suggestive of a similar enzymatic mechanism. RlmO homologues are primarily found in mesophilic bacteria related to T. thermophilus. In accordance, we find that growth of the T. thermophilus strain with an inactivated C1942 methyltransferase gene is not compromised at non-optimal temperatures.

Distinction between the Cfr methyltransferase conferring antibiotic resistance and the housekeeping RlmN methyltransferase.

The cfr gene encodes the Cfr methyltransferase that primarily methylates C-8 in A2503 of 23S rRNA in the peptidyl transferase region of bacterial ribosomes. The methylation provides resistance to six classes of antibiotics of clinical and veterinary importance. The rlmN gene encodes the RlmN methyltransferase that methylates C-2 in A2503 in 23S rRNA and A37 in tRNA, but RlmN does not significantly influence antibiotic resistance. The enzymes are homologous and use the same mechanism involving radical S-adenosyl methionine to methylate RNA via an intermediate involving a methylated cysteine in the enzyme and a transient cross-linking to the RNA, but they differ in which carbon atom in the adenine they methylate. Comparative sequence analysis identifies differentially conserved residues that indicate functional sequence divergence between the two classes of Cfr- and RlmN-like sequences. The differentiation between the two classes is supported by previous and new experimental evidence from antibiotic resistance, primer extensions, and mass spectrometry. Finally, evolutionary aspects of the distribution of Cfr- and RlmN-like enzymes are discussed.

Roles of Trm9- and ALKBH8-like proteins in the formation of modified wobble uridines in Arabidopsis tRNA.

Uridine at the wobble position of tRNA is usually modified, and modification is required for accurate and efficient protein translation. In eukaryotes, wobble uridines are modified into 5-methoxycarbonylmethyluridine (mcm(5)U), 5-carbamoylmethyluridine (ncm(5)U) or derivatives thereof. Here, we demonstrate, both by in vitro and in vivo studies, that the Arabidopsis thaliana methyltransferase AT1G31600, denoted by us AtTRM9, is responsible for the final step in mcm(5)U formation, thus representing a functional homologue of the Saccharomyces cerevisiae Trm9 protein. We also show that the enzymatic activity of AtTRM9 depends on either one of two closely related proteins, AtTRM112a and AtTRM112b. Moreover, we demonstrate that AT1G36310, denoted AtALKBH8, is required for hydroxylation of mcm(5)U to (S)-mchm(5)U in tRNA(Gly)(UCC), and has a function similar to the mammalian dioxygenase ALKBH8. Interestingly, atalkbh8 mutant plants displayed strongly increased levels of mcm(5)U, and also of mcm(5)Um, its 2'-O-ribose methylated derivative. This suggests that accumulated mcm(5)U is prone to further ribose methylation by a non-specialized mechanism, and may challenge the notion that the existence of mcm(5)U- and mcm(5)Um-containing forms of the selenocysteine-specific tRNA(Sec) in mammals reflects an important regulatory process. The present study reveals a role in for several hitherto uncharacterized Arabidopsis proteins in the formation of modified wobble uridines.

Multi-site-specific 16S rRNA methyltransferase RsmF from Thermus thermophilus.

Cells devote a significant effort toward the production of multiple modified nucleotides in rRNAs, which fine tune the ribosome function. Here, we report that two methyltransferases, RsmB and RsmF, are responsible for all four 5-methylcytidine (m(5)C) modifications in 16S rRNA of Thermus thermophilus. Like Escherichia coli RsmB, T. thermophilus RsmB produces m(5)C967. In contrast to E. coli RsmF, which introduces a single m(5)C1407 modification, T. thermophilus RsmF modifies three positions, generating m(5)C1400 and m(5)C1404 in addition to m(5)C1407. These three residues are clustered near the decoding site of the ribosome, but are situated in distinct structural contexts, suggesting a requirement for flexibility in the RsmF active site that is absent from the E. coli enzyme. Two of these residues, C1400 and C1404, are sufficiently buried in the mature ribosome structure so as to require extensive unfolding of the rRNA to be accessible to RsmF. In vitro, T. thermophilus RsmF methylates C1400, C1404, and C1407 in a 30S subunit substrate, but only C1400 and C1404 when naked 16S rRNA is the substrate. The multispecificity of T. thermophilus RsmF is potentially explained by three crystal structures of the enzyme in a complex with cofactor S-adenosyl-methionine at up to 1.3 A resolution. In addition to confirming the overall structural similarity to E. coli RsmF, these structures also reveal that key segments in the active site are likely to be dynamic in solution, thereby expanding substrate recognition by T. thermophilus RsmF.

Platinum interference with siRNA non-seed regions fine-tunes silencing capacity.

Knowledge concerning the molecular mechanisms governing the influence of non-coding RNAs on protein production has emerged rapidly during the past decade. Today, two main research areas can be identified, one oriented toward the use of artificially introduced siRNAs for manipulation of gene expression, and the other one focused on the function of endogenous miRNAs. In both cases, the active molecule consists of a ∼20-nucleotide-long RNA duplex. In the siRNA case, improved systemic stability is of central interest for its further development toward clinical applications. With respect to miRNA processing and function, understanding its influence on mRNA targeting and the silencing ability of individual miRNAs, e.g., under pathological conditions, remains a scientific challenge. In the present study, a model system is presented where the influence of the two clinically used anticancer drugs, cisplatin and oxaliplatin, on siRNA's silencing capacity has been evaluated. More specifically, siRNAs targeting the 3' UTR region of Wnt-5a mRNA (NM_003352) were constructed, and the biologically active antisense RNA strand was pre-platinated. Platinum adducts were detected and characterized by a combination of gel electrophoresis and MALDI-MS techniques, and the silencing capacity was evaluated in cellular luciferase-expressing systems using HB2 cells. Data show that platination of the antisense strand of the siRNAs results in adducts with protection against hydrolytic cleavage in the proximity of the platination sites, i.e., with altered degradation patterns compared to native RNAs. The MALDI-MS method was successfully used to further identify and characterize platinated RNA, with the naturally occurring platinum isotopic patterns serving as sensitive fingerprints for metalated sites. Expression assays all confirm biological activity of antisense-platinated siRNAs, here with platination sites located outside of the seed region. A significant reduction of silencing capacity was observed as a general trend, however. Of the two complexes studied, oxaliplatin exhibits the larger influence, thus indicating subtle differences between the abilities of cis- and oxaliplatin to interfere with si- and miRNA processing.

Minimal substrate features for Erm methyltransferases defined by using a combinatorial oligonucleotide library.

Erm methyltransferases are prevalent in pathogenic bacteria and confer resistance to macrolide, lincosamide, and streptogramin B antibiotics by specifically methylating the 23S ribosomal RNA at nucleotide A2058. We have identified motifs within the rRNA substrate that are required for methylation by Erm. Substrate molecules were constructed in a combinatorial manner from two separate sets (top and bottom strands) of short RNA sequences. Modifications, including LNA monomers with locked sugar residues, were incorporated into the substrates to stabilize their structures. In functional substrates, the A2058 methylation target (on the 13- to 19-nucleotide top strand) was displayed in an unpaired sequence immediately following a conserved irregular helix, and these are the specific structural features recognized by Erm. Erm methylation was enhanced by stabilizing the top-strand conformation with an LNA residue at G2056. The bottom strand (nine to 19 nucleotides in length) was required for methylation and was still functional after extensive modification, including substitution with a DNA sequence. Although it remains possible that Erm makes some unspecific contact with the bottom strand, the main role played by the bottom strand appears to be in maintaining the conformation of the top strand. The addition of multiple LNA residues to the top strand impeded methylation; this indicates that the RNA substrate requires a certain amount of flexibility for accommodation into the active site of Erm. The combinatorial approach for identifying small but functional RNA substrates is a step towards making RNA-Erm complexes suitable for cocrystal determination, and for designing molecules that might block the substrate-recognition site of the enzyme.

Identification of 8-methyladenosine as the modification catalyzed by the radical SAM methyltransferase Cfr that confers antibiotic resistance in bacteria.

The Cfr methyltransferase confers combined resistance to five different classes of antibiotics that bind to the peptidyl transferase center of bacterial ribosomes. The Cfr-mediated modification has previously been shown to occur on nucleotide A2503 of 23S rRNA and has a mass corresponding to an additional methyl group, but its specific identity and position remained to be elucidated. A novel tandem mass spectrometry approach has been developed to further characterize the Cfr-catalyzed modification. Comparison of nucleoside fragmentation patterns of A2503 from Escherichia coli cfr+ and cfr- strains with those of a chemically synthesized nucleoside standard shows that Cfr catalyzes formation of 8-methyladenosine. In addition, analysis of RNA derived from E. coli strains lacking the m(2)A2503 methyltransferase reveals that Cfr also has the ability to catalyze methylation at position 2 to form 2,8-dimethyladenosine. The mutation of single conserved cysteine residues in the radical SAM motif CxxxCxxC of Cfr abolishes its activity, lending support to the notion that the Cfr modification reaction occurs via a radical-based mechanism. Antibiotic susceptibility data confirm that the antibiotic resistance conferred by Cfr is provided by methylation at the 8 position and is independent of methylation at the 2 position of A2503. This investigation is, to our knowledge, the first instance where the 8-methyladenosine modification has been described in natural RNA molecules.

Identification of RNA molecules by specific enzyme digestion and mass spectrometry: software for and implementation of RNA mass mapping.

The idea of identifying or characterizing an RNA molecule based on a mass spectrum of specifically generated RNA fragments has been used in various forms for well over a decade. We have developed software-named RRM for 'RNA mass mapping'-which can search whole prokaryotic genomes or RNA FASTA sequence databases to identify the origin of a given RNA based on a mass spectrum of RNA fragments. As input, the program uses the masses of specific RNase cleavage of the RNA under investigation. RNase T1 digestion is used here as a demonstration of the usability of the method for RNA identification. The concept for identification is that the masses of the digestion products constitute a specific fingerprint, which characterize the given RNA. The search algorithm is based on the same principles as those used in peptide mass fingerprinting, but has here been extended to work for both RNA sequence databases and for genome searches. A simple and powerful probability model for ranking RNA matches is proposed. We demonstrate viability of the entire setup by identifying the DNA template of a series of RNAs of biological and of in vitro transcriptional origin in complete microbial genomes and by identifying authentic 16S ribosomal RNAs in a 'small ribosomal subunit RNA' database. Thus, we present a new tool for a rapid identification of unknown RNAs using only a few picomoles of starting material.

Intrinsic Strand-Incision Activity of Human UNG: Implications for Nick Generation in Immunoglobulin Gene Diversification.

Uracil arises in cellular DNA by cytosine (C) deamination and erroneous replicative incorporation of deoxyuridine monophosphate opposite adenine. The former generates C → thymine transition mutations if uracil is not removed by uracil-DNA glycosylase (UDG) and replaced by C by the base excision repair (BER) pathway. The primary human UDG is hUNG. During immunoglobulin gene diversification in activated B cells, targeted cytosine deamination by activation-induced cytidine deaminase followed by uracil excision by hUNG is important for class switch recombination (CSR) and somatic hypermutation by providing the substrate for DNA double-strand breaks and mutagenesis, respectively. However, considerable uncertainty remains regarding the mechanisms leading to DNA incision following uracil excision: based on the general BER scheme, apurinic/apyrimidinic (AP) endonuclease (APE1 and/or APE2) is believed to generate the strand break by incising the AP site generated by hUNG. We report here that hUNG may incise the DNA backbone subsequent to uracil excision resulting in a 3´-α,ß-unsaturated aldehyde designated uracil-DNA incision product (UIP), and a 5´-phosphate. The formation of UIP accords with an elimination (E2) reaction where deprotonation of C2´ occurs via the formation of a C1´ enolate intermediate. UIP is removed from the 3´-end by hAPE1. This shows that the first two steps in uracil BER can be performed by hUNG, which might explain the significant residual CSR activity in cells deficient in APE1 and APE2.