RNA ribose methylation (2′-O-methylation): Occurrence,biosynthesis and biological functions

Methylation of riboses at 2′-OH group is one of the most common RNA modifications found in number of cellular RNAs from almost any species which belong to all three life domains. This modification was extensively studied for decades in rRNAs and tRNAs, but recent data revealed the presence of 2′-O-methyl groups also in low abundant RNAs, like mRNAs.Ribose methylation is formed in RNA by two alternative enzymatic mechanisms: either by stand-alone protein enzymes or by complex assembly of proteins associated with snoRNA guides (sno(s)RNPs). In that case one catalytic subunit acts at various RNA sites, the specificity is provided by base pairing of the sno(s)RNA guide with the target RNA. In this review we compile available information on 2′-OH ribose methylation in different RNAs, enzymatic machineries involved in their biosynthesis and dynamics, as well as on the physiological functions of these modified residues.     

Nucleobase catalysis in the hairpin ribozyme

RNA catalysis is important in the processing and translation of RNA molecules, yet the mechanisms of catalysis are still unclear in most cases. We have studied the role of nucleobase catalysis in the hairpin ribozyme, where the scissile phosphate is juxtaposed between guanine and adenine bases. We show that a modified ribozyme in which guanine 8 has been substituted by an imidazole base is active in both cleavage and ligation, with ligation rates 10-fold faster than cleavage. The rates of both reactions exhibit bell-shaped dependence on pH, with pK(a) values of 5.7 +/- 0.1 and 7.7 +/- 0.1 for cleavage and 6.1 +/- 0.3 and 6.9 +/- 0.3 for ligation. The data provide good evidence for general acid-base catalysis by the nucleobases.     

A systematic, ligation-based approach to study RNA modifications

Over 100 different chemical types of modifications have been identified in thousands of sites in tRNAs, rRNAs, mRNAs, small nuclear RNAs, and other RNAs. Some modifications are highly conserved, while others are more specialized. They include methylation of bases and the ribose backbone, rotation, and reduction of uridine, base deamination, elaborate addition of ring structures, carbohydrate moieties, and more. We have developed a systematic approach to detect and quantify the extent of known RNA modifications. The method is based on the enzymatic ligation of oligonucleotides using the modified or unmodified RNA as the template. The efficiency of ligation is very sensitive to the presence and the type of modifications. First, two oligo pairs for each type of modification are identified. One pair greatly prefers ligation using the unmodified RNA template over the modified RNA template or vice versa. The other pair has equal reactivity with unmodified and modified RNA. Second, separate ligations with each of the two oligo pairs and the total RNA mixture are performed to detect the presence or absence of modifications. Multiple modification sites can be examined in the same ligation reaction. The feasibility of this method is demonstrated for three 2'O-methyl modification sites in yeast rRNA.     

The effect of structure in a long target RNA on ribozyme cleavage efficiency.

Inhibition of gene expression by catalytic RNA (ribozymes) requires that ribozymes efficiently cleave specific sites within large target RNAs. However, the cleavage of long target RNAs by ribozymes is much less efficient than cleavage of short oligonucleotide substrates because of higher order structure in the long target RNA. To further study the effects of long target RNA structure on ribozyme cleavage efficiency, we determined the accessibility of seven hammerhead ribozyme cleavage sites in a target RNA that contained human immunodeficiency virus type 1 (HIV-1) vif - vpr . The base pairing-availability of individual nucleotides at each cleavage site was then assessed by chemical modification mapping. The ability of hammerhead ribozymes to cleave the long target RNA was most strongly correlated with the availability of nucleotides near the cleavage site for base pairing with the ribozyme. Moreover, the accessibility of the seven hammerhead ribozyme cleavage sites in the long target RNA varied by up to 400-fold but was directly determined by the availability of cleavage sites for base pairing with the ribozyme. It is therefore unlikely that steric interference affected hammerhead ribozyme cleavage. Chemical modification mapping of cleavage site structure may therefore provide a means to identify efficient hammerhead ribozyme cleavage sites in long target RNAs.     

A chemical interference study on the interaction of ribosomal protein L11 from Escherichia coli with RNA molecules containing its binding site from 23S rRNA.

The interaction between ribosomal protein L11 from Escherichia coli and in vitro synthesized RNA containing its binding site from 23S rRNA was characterized by identifying nucleotides that interfered with complex formation when chemically modified by diethylpyrocarbonate or hydrazine. Chemically modified RNA was incubated with L11 under conditions appropriate for specific binding of L11 and the resulting protein-RNA complex was separated from unbound RNA on Mg(2+)-containing polyacrylamide gels. The ability to isolate L11 complexes on such gels was affected by the extent of modification by either reagent. Protein-bound and free RNAs were recovered and treated with aniline to identify their content of modified bases. Exclusion of RNA containing chemically altered bases from L11-associated material occurred for 29 modified nucleotides, located throughout the region corresponding to residues 1055-1105 in 23S rRNA. Ten bases within this region did not reproducibly inhibit binding when modified. Multiple bands of RNA were consistently observed on the nondenaturing gels, suggesting that significant intermolecular RNA-RNA interactions had occurred.     

Phylogenetic comparative chemical footprint analysis of the interaction between ribonuclease P RNA and tRNA.

Ribonuclease P RNA is the catalytic moiety of the ribonucleoprotein enzyme that endonucleolytically cleaves precursor sequences from the 5' ends of pre-tRNAs. The bacterial RNase P RNA-tRNA complex was examined with a footprinting approach, utilizing chemical modification to determine RNase P RNA nucleotides that potentially contact tRNA. RNase P RNA was modified with dimethylsulfate or kethoxal in the presence or absence of tRNA, and sites of modification were detected by primer extension. Comparison of the results reveals RNase P bases that are protected from modification upon binding tRNA. Analyses were carried out with RNase P RNAs from three different bacteria: Escherichia coli, Chromatium vinosum and Bacillus subtilis. Discrete bases of these RNAs that lie within conserved, homologous portions of the secondary structures are similarly protected. One protection among all three RNAs was attributed to the precursor segment of pre-tRNA. Experiments using pre-tRNAs containing precursor segments of variable length demonstrate that a precursor segment of only 2-4 nucleotides is sufficient to confer this protection. Deletion of the 3'-terminal CCA sequence of tRNA correlates with loss of protection of a particular loop in the RNase P RNA secondary structure. Analysis of mutant tRNAs containing sequential 3'-terminal deletions suggests a relative orientation of the bound tRNA CCA to that loop.     

Effects of the ribosomal subunit association on the chemical modification of the 16S and 23S RNAs from Escherichia coli

70S ribosomes and 30S and 50S ribosomal subunits from Escherichia coli were modified under non-denaturing conditions with the chemical reagent dimethylsulfate. The ribosomal 23S and 16S RNAs were isolated after the reaction and the last 200 nucleotides from the 3' ends were analyzed for differences in the chemical modification. A number of accessibility changes could be detected for 23S and 16S RNA when 70S ribosomes as opposed to the isolated subunits were modified. In addition to a number of sites which were protected from modification several guanosines showed enhanced reactivities, indicating conformational changes in the ribosomal RNA structures when 30S and 50S subunits associate to a 70S particle. Most of the accessibility changes can be localized in double-helical regions within the secondary structures of the two RNAs. The results confirm the importance of the ribosomal RNAs for ribosomal functions and help to define the RNA domains which constitute the subunit interface of E. coli ribosomes.     

Substrate recognition by RNase P and by the catalytic M1 RNA: identification of possible contact points in pre-tRNAs.

Modified bases were introduced into pre-tRNAs during in vitro RNA synthesis or by chemical modification. These RNAs were used as substrates for the catalytic M1 RNA and the RNase P holoenzyme from Schizosaccharomyces pombe. The synthetic approach permitted the insertion of 100% m7GTP into pre-tRNAs and this resulted in complete inhibition of the specific 5' processing reactions. Partially modified RNAs were obtained by chemical modifications of purines and uridines in the pre-tRNAs. This allowed detailed analyses of specific bases excluded in the products. With pre-tRNA(Ser) and initiator pre-tRNA(Met), strong effects were observed in the T arm and weaker effects in the anticodon stem. Only minor base exclusions were detected in the acceptor stem of pre-tRNA(Ser) and in the D arm of pre-tRNA(Met).     

Introduction: Metals in Biology: α-Ketoglutarate/Iron-Dependent Dioxygenases

Four minireviews deal with aspects of the α-ketoglutarate/iron-dependent dioxygenases in this eighth Thematic Series on Metals in Biology. The minireviews cover a general introduction and synopsis of the current understanding of mechanisms of catalysis, the roles of these dioxygenases in post-translational protein modification and de-modification, the roles of the ten-eleven translocation (Tet) dioxygenases in the modification of methylated bases (5mC, T) in DNA relevant to epigenetic mechanisms, and the roles of the AlkB-related dioxygenases in the repair of damaged DNA and RNA. The use of α-ketoglutarate (alternatively termed 2-oxoglutarate) as a co-substrate in so many oxidation reactions throughout much of nature is notable and has surprisingly emerged from biochemical and genomic analysis. About 60 of these enzymes are now recognized in humans, and a number have been identified as having critical functions.     

The three-dimensional folding of the tRNA-like structure of tobacco mosaic virus RNA. A new building principle applied twice

The structure of the tRNA-like 3' terminus of tobacco mosaic virus (TMV) RNA has been studied. A 3' -terminal fragment possessing the tRNA-like properties was probed with chemical modification and enzymatic digestions. A model of the secondary structure is proposed for the last 105 nucleotides. The corresponding region of other tobamoviral RNAs can be folded in an identical secondary structure. A three-dimensional model for the tRNA-like structure is given which is compared with those proposed earlier for the tRNA-like 3' termini of turnip yellow mosaic virus (TYMV) RNA and brome mosaic virus (BMV) RNA. A new building principle which we discovered previously by studying the latter RNAs appears to be applied twice in the tRNA-like structure of TMV RNA. The determination of the minimal length requirement for recognition of CTP, ATP:tRNA nucleotidyl-transferase reveals a size of ˜100 nucleotides in agreement with the models proposed.     

A Metazoan/Plant-like Capping Enzyme and Cap Modified Nucleotides in the Unicellular Eukaryote Trichomonas vaginalis

The cap structure of eukaryotic messenger RNAs is initially elaborated through three enzymatic reactions: hydrolysis of the 5′-triphosphate, transfer of guanosine through a 5′-5′ triphosphate linkage and N7-methylation of the guanine cap. Three distinctive enzymes catalyze each reaction in various microbial eukaryotes, whereas the first two enzymes are fused into a single polypeptide in metazoans and plants. In addition to the guanosine cap, adjacent nucleotides are 2′-O-ribose methylated in metazoa and plants, but not in yeast. Analyses of various cap structures have suggested a linear phylogenetic trend of complexity. These findings have led to a model in which plants and metazoa evolved a two-component capping apparatus and modification of adjacent nucleotides while many microbial eukaryotes maintained the three-component system and did not develop modification of adjacent nucleotides. Here, we have characterized a bifunctional capping enzyme in the divergent microbial eukaryote Trichomonas vaginalis using biochemical and phylogenetic analyses. This unicellular parasite was found to harbor a metazoan/plant-like capping apparatus that is represented by a two-domain polypeptide containing a C-terminus guanylyltransferase and a cysteinyl phosphatase triphosphatase, distinct from its counterpart in other microbial eukaryotes. In addition, T. vaginalis mRNAs contain a cap 1 structure represented by m⁷GpppAmpUp or m⁷GpppCmpUp; a feature typical of metazoan and plant mRNAs but absent in yeast mRNAs. Phylogenetic and biochemical analyses of the origin of the T. vaginalis capping enzyme suggests a complex evolutionary model where differential gene loss and/or acquisition occurred in the development of the RNA capping apparatus and cap modified nucleotides during eukaryote diversification.     

Genotoxicity of singlet oxygen

Singlet oxygen, ¹O₂(¹Δ_g), fulfills essential prerequisites for a genotoxic substance, like hydroxyl radicals and other oxygen radicals: it can react efficiently with DNA and it can be generated inside cells, e.g. by photosensitization and enzymatic oxidation. As might be anticipated from the non-radical character of singlet oxygen, the pattern of DNA modifications it produces is very different from that caused by hydroxyl radicals. While hydroxyl radicals produce DNA strand breaks and sites of base loss (AP sites) in high yield and react with all four bases of DNA, singlet oxygen generates predominantly modified guanine residues and few strand breaks and AP sites. There is now convincing evidence that a major product of base modification caused by singlet oxygen is 8-hydroxyguanine (7,8-dihydro-8-oxoguanine). Indeed, the recently reported miscoding properties of 8-hydroxyguanine can explain the predominant type of mutations observed when DNA modified by singlet oxygen is replicated in cells. There are also strong indications that singlet oxygen generated by photosensitization can act as an ultimate DNA modifying species inside cells. However, indirect genotoxic mechanisms involving other reactive oxygen species produced from singlet oxygen are also possible and appear to predominate in some cases. The cellular defense system against oxidants consists of effective singlet oxygen scavengers such as carotenoids. The observation that carotenoids can inhibit neoplastic cell transformation when administered not only together with but also after the application of chemical or physical carcinogens might indicate a role of singlet oxygen in tumor promotion that could be independent of the direct or indirect DNA damaging properties.     

Five pseudoknots are present at the 204 nucleotides long 3'' noncoding region of tobacco mosaic virus RNA.

The 104 nucleotides long 3' terminal region of TMV RNA was shown previously to contain two pseudoknotted structures (Rietveld et al. (1984), EMBO J. 3, 2613-2619). We here present evidence for the occurrence, within the 204 nucleotides long 3' noncoding region, of another highly structured domain located immediately adjacent to the tRNA-like structure of 95 nucleotides (Joshi et al. (1985) Nucleic Acids Res. 13, 347-354). A model for the three-dimensional folding of this region, containing three more pseudoknots, is proposed on the basis of chemical modification and enzymatic digestion. The existence of these three consecutive pseudoknots was supported by sequence comparisons with the RNA from the related tobamoviruses TMV-L, CcTMV and CGMMV. Coaxial stacking of the six double helical segments involved gives rise to the formation of a 25 basepair long quasi-continuous double helix. The results show that the three-dimensional folding of the 3' non-translated region of tobamoviral RNAs is largely maintained by the formation of five pseudoknots. The organisation of this region in the RNA of the tobamovirus CcTMV suggests that recombinational events among aminoacylatable plant viral RNAs have to be considered.     

Base-specific reactions useful for DNA sequencing: methylene blue--sensitized photooxidation of guanine and osmium tetraoxide modification of thymine.

Exposure of DNA to methylene blue and visible or ultraviolet light causes guanine-specific modification, and subsequent treatment with piperidine leads to chain cleavage at each guanine residue. Treatment of DNA with osmium tetraoxide in dilute pyridine leads to thymidine-specific modification, and subsequent treatment with piperidine leads to chain cleavage at the modified thymidine residues. Both reactions can be used in conjunction with other base specific modifications described by Maxam and Gilbert (1) for the determination of the nucleotide sequence in DNA.