Improved application of RNAModMapper - An RNA modification mapping software tool - For analysis of liquid chromatography tandem mass spectrometry (LC-MS/MS) data.

Research into post-transcriptional processing and modification of RNA continues to speed forward, as their ever-emerging role in the regulation of gene expression in biological systems continues to unravel. Liquid chromatography tandem mass spectrometry (LC-MS/MS) has proven for over two decades to be a powerful ally in the elucidation of RNA modification identity and location, but the technique has not proceeded without its own unique technical challenges. The throughput of LC-MS/MS modification mapping experiments continues to be impeded by tedious and time-consuming spectral interpretation, particularly during for the analysis of complex RNA samples. RNAModMapper was recently developed as a tool to improve the interpretation and annotation of LC-MS/MS data sets from samples containing post-transcriptionally modified RNAs. Here, we delve deeper into the methodology and practice of RNAModMapper to provide greater insight into its utility, and remaining hurdles, in current RNA modification mapping experiments.

1H, 15N chemical shift assignments of the imino groups of yeast tRNAPhe: influence of the post-transcriptional modifications.

Transfer RNAs (tRNAs) are heavily decorated with post-transcriptional modifications during their biosynthesis. To fulfil their functions within cells, tRNAs undergo a tightly controlled biogenesis process leading to the formation of mature tRNAs. In particular, the introduction of post-transcriptional modifications in tRNAs is controlled and influenced by multiple factors. In turn, tRNA biological functions are often modulated by their modifications. Although modifications play essential roles in tRNA biology, methods to directly detect their introduction during tRNA maturation are rare and do not easily provide information on the temporality of modification events. To obtain information on the tRNA maturation process, we have developed a methodology, using NMR as a tool to monitor tRNA maturation in a non-disruptive and continuous fashion in cellular extracts. Here we report the 1H,15N chemical shift assignments of imino groups in three forms of the yeast tRNAPhe differing in their modification content. These assignments are a prerequisite for the time-resolved NMR monitoring of yeast tRNAPhe maturation in yeast extracts.

Defining the inside and outside of a catalytic RNA molecule.

Ribozymes are RNA molecules that catalyze biochemical reactions. Fe(II)-EDTA, a solvent-based reagent which cleaves both double- and single-stranded RNA, was used to investigate the structure of the Tetrahymena ribozyme. Regions of cleavage alternate with regions of substantial protection along the entire RNA molecule. In particular, most of the catalytic core shows greatly reduced cleavage. These data constitute experimental evidence that an RNA enzyme, like a protein enzyme, has an interior and an exterior. Determination of positions where the phosphodiester backbone of the RNA is on the inside or on the outside of the molecule provides major constraints for modeling the three-dimensional structure of the Tetrahymena ribozyme. This approach should be generally informative for structured RNA molecules.

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.

Transfer RNA identity change in anticodon variants of E. coli tRNA(Phe) in vivo.

The anticodon sequence is a major recognition element for most aminoacyl-tRNA synthetases. We investigated the in vivo effects of changing the anticodon on the aminoacylation specificity in the example of E. coli tRNA(Phe). Constructing different anticodon mutants of E. coli tRNA(Phe) by site-directed mutagenesis, we isolated 22 anticodon mutant tRNA(Phe); the anticodons corresponded to 16 amino acids and an opal stop codon. To examine whether the mutant tRNAs had changed their amino acid acceptor specificity in vivo, we tested the viability of E. coli strains containing these tRNA(Phe) genes in a medium which permitted tRNA induction. Fourteen mutant tRNA genes did not affect host viability. However, eight mutant tRNA genes were toxic to the host and prevented growth, presumably because the anticodon mutants led to translational errors. Many mutant tRNAs which did not affect host viability were not aminoacylated in vivo. Three mutant tRNAs containing anticodon sequences corresponding to lysine (UUU), methionine (CAU) and threonine (UGU) were charged with the amino acid corresponding to their anticodon, but not with phenylalanine. These three tRNAs and tRNA(Phe) are located in the same cluster in a sequence similarity dendrogram of total E. coli tRNAs. The results support the idea that such tRNAs arising from in vivo evolution are derived by anticodon change from the same ancestor tRNA.

Evidence for RNA in the peptidyl transferase center of Escherichia coli ribosomes as indicated by fluorescence.

A coumarin derivative was covalently attached to either the amino acid or the 5' end of phenylalanine-specific transfer RNA (tRNA(phe)). Its fluorescence was quenched by methyl viologen when the tRNA was free in solution or bound to Escherichia coli ribosomes. Methyl viologen as a cation in solution has a strong affinity for the ionized phosphates of a nucleic acid and so can be used to qualitatively measure the presence of RNA in the immediate vicinity of the tRNA-linked coumarins upon binding to ribosomes. Fluorescence lifetime measurements indicate that the increase in fluorescence quenching observed when the tRNAs are bound into the peptidyl site of ribosomes is due to static quenching by methyl viologen bound to RNA in the immediate vicinity of the fluorophore. The data lead to the conclusion that the ribosome peptidyl transferase center is rich in ribosomal RNA. Movement of the fluorophore at the N-terminus of the nascent peptide as it is extended or movement of the tRNA acceptor stem away from the peptidyl transferase center during peptide bond formation appears to result in movement of the probe into a region containing less rRNA.

Visualization of RNA tertiary structure by RNA-EDTA.Fe(II) autocleavage: analysis of tRNA(Phe) with uridine-EDTA.Fe(II) at position 47.

To test whether intramolecular autocleavage of RNA labeled at a single nucleotide position with EDTA.Fe(II) is a useful approach for the investigation of tertiary structures of RNAs, yeast phenylalanine tRNA was synthesized with uridine-EDTA (*U) at position U47. Autocleavage of [*U47]tRNA(Phe) in the presence of Fe(NH4)2(SO4)2 and dithiothreitol produced a set of cleavage fragments which are in general agreement with the three-dimensional structure derived from x-ray analysis.

High resolution chromatography of ribonucleosides and its application to RNA analysis.

A simple and precise method was developed for the separation of nucleosides including modified nucleosides and oligonucleotides. Nineteen kinds of nucleosides were completely separated by HPLC using an ODS column (TSK-gel ODS 80TM) and aqueous mobile phases. The RNA molecule was digested by base restrictive RNase (RNase A, RNase T1) and the digests were separated chromatographically into each oligonucleotide. The nucleoside composition of an oligonucleotide was then determined by this analytical system. It is thus possible to fit the oligonucleotide in the original RNA molecule by using modified bases as markers. The reaction site of quinacrine mustard for tRNA(Phe) (from yeast) could be determined by this analytical system.

Probing the environment of lanthanide binding sites in yeast tRNA(Phe) by specific metal-ion-promoted cleavages.

Specific yeast tRNA(Phe) hydrolysis brought about by europium ions has been studied in detail using the 32P-end-labeled tRNA and polyacrylamide gel electrophoresis. The dependence of the induced cleavages on pH, temperature and concentration of the europium ions has been determined. Europium hydrolyzes yeast tRNA(Phe) in the D-loop at phosphates 16 and 18, and the anticodon loop of phosphates 34 and 36. The two D-loop cuts are thought to take place from two distinct europium binding sites, while the two anticodon loop cleavages from a single site. Eight other members of the lanthanide series and ytrium give basically the same pattern of cleavages as europium. The specific cleavages taking place in the anticodon loop occur in an intramolecular mode from the lanthanide binding site that has not been found in yeast tRNA(Phe) crystal structure. It appears from the comparison of the europium-promoted cuts with those generated by magnesium and lead that the former two ions give more similar but not identical cleavage patterns. The usefulness of the specific cleavages induced by lanthanides for probing their own and magnesium binding sites in tRNA is discussed.

Nonradioactive labeling of RNA.

A modified ribodinucleotide (named pSEEp) has been synthesized using commercially available components on a DNA synthesizer. The presence of a 3'-terminal primary amino group permits its coupling to a range of nonradioactive labels, exemplified here by fluorescein. The product of chemical derivatization of the parent dinucleotide is a good substrate for T4 RNA ligase-mediated coupling to RNA under very mild conditions; the target RNA is not itself subjected to chemical treatment. The well-defined product of this terminal labeling may be analyzed by, for example, the use of a fluorescence-based DNA sequencer. The applications documented serve to demonstrate the power of this approach suggesting that any procedure depending on the detection or targeted tagging of RNA may be adapted to using pSEEp and its derivatives.

tRNA discrimination by T. thermophilus phenylalanyl-tRNA synthetase at the binding step.

The extent of tRNA recognition at the level of binding by Thermus thermophilus phenylalanyl-tRNA synthetase (PheRS), one of the most complex class II synthetases, has been studied by independent measurements of the enzyme association with wild-type and mutant tRNA(Phe)s as well as with non-cognate tRNAs. The data obtained, combined with kinetic data on aminoacylation, clearly show that PheRS exhibits more tRNA selectivity at the level of binding than at the level of catalysis. The anticodon nucleotides involved in base-specific interactions with the enzyme prevail both in the initial binding recognition and in favouring aminoacylation catalysis. Tertiary nucleotides of base pair G19-C56 and base triple U45-G10-C25 contribute primarily to stabilization of the correctly folded tRNA(Phe) structure, which is important for binding. Other nucleotides of the central core (U20, U16 and of the A26-G44 tertiary base pair) are involved in conformational adjustment of the tRNA upon its interaction with the enzyme. The specificity of nucleotide A73, mutation of which slightly reduces the catalytic rate of aminoacylation, is not displayed at the binding step. A few backbone-mediated contacts of PheRS with the acceptor and anticodon stems revealed in the crystal structure do not contribute to tRNA(Phe) discrimination, their role being limited to stabilization of the complex. The highest affinity of T. thermophilus PheRS for cognate tRNA, observed for synthetase-tRNA complexes, results in 100-3000-fold binding discrimination against non-cognate tRNAs.

Analysis of magnesium, europium and lead binding sites in methionine initiator and elongator tRNAs by specific metal-ion-induced cleavages.

The specificity of cleavages in yeast and lupin initiator and elongator methionine tRNAs induced by magnesium, europium and lead has been analysed and compared with known patterns of yeast tRNA(Phe) hydrolysis. The strong D-loop cleavages occur in methionine elongator tRNAs at similar positions and with comparable efficiency to those found in tRNA(Phe), while the sites of weak anticodon loop cuts, identical in methionine elongator tRNAs, differ from those found in tRNA(Phe). Methionine initiator tRNAs differ from their elongator counterparts: (a) they are cleaved in the D-loop with much lower efficiency; (b) they are cleaved in the variable loop which is completely resistant to hydrolysis in elongator tRNAs; (c) cleavages in the anticodon loop are stronger in initiator tRNAs and they are located mostly at the 5' side of the loop whereas in elongator tRNAs they occur mostly at the opposite, 3' side of the loop. The distinct pattern of the anticodon loop cleavages is considered to be related to different conformations of the anticodon loop in the two types of methionine tRNAs.