RNA Modification Mapping Research Articles

Mapping of Posttranscriptional tRNA Modifications by Two-Dimensional Gel Electrophoresis Mass Spectrometry.

RNA modification mapping by mass spectrometry (MS) is based on the use of specific ribonucleases (RNases) that generate short oligonucleotide digestion products which are further separated by nano-liquid chromatography and analyzed by MS and MS/MS. Recent developments in MS instrumentation allow the possibility to deeply explore posttranscriptional modifications. Notably, development of nano-liquid chromatography and nano-electrospray drastically increases the detection sensitivity and allows the identification and sequencing of RNA digested fragments separated and extracted from two-dimensional polyacrylamide gels, as long as the mapping and characterization of ribonucleotide modifications.

Improved RNA modification mapping of cellular non-coding RNAs using C- and U-specific RNases

Locating ribonucleoside modifications within an RNA sequence requires digestion of the RNA into oligoribonucleotides of amenable size for subsequent analysis by LC-MS (liquid chromatography-mass spectrometry). This approach, widely referred to as RNA modification mapping, is facilitated through ribonucleases (RNases) such as T1 (guanosine-specific), U2 (purine-selective) and A (pyrimidine-specific) among others. Sequence coverage by these enzymes depends on positioning of the recognized nucleobase (such as guanine or purine or pyrimidine) in the sequence and its ribonucleotide composition. Using E. coli transfer RNA (tRNA) and ribosomal RNA (rRNA) as model samples, we demonstrate the ability of complementary nucleobase-specific ribonucleases cusativin (C-specific) and MC1 (U-specific) to generate digestion products that facilitate confident mapping of modifications in regions such as G-rich and pyrimidine-rich segments of RNA, and to distinguish C to U sequence differences. These enzymes also increase the number of oligonucleotide digestion products that are unique to a specific RNA sequence. Further, with these additional RNases, multiple modifications can be localized with high confidence in a single set of experiments with minimal dependence on the individual tRNA abundance in a mixture. The sequence overlaps observed with these complementary digestion products and that of RNase T1 improved sequence coverage to 75% or above. A similar level of sequence coverage was also observed for the 2904 nt long 23S rRNA indicating their utility has no dependence on RNA size. Wide-scale adoption of these additional modification mapping tools could help expedite the characterization of modified RNA sequences to understand their structural and functional role in various living systems.

Using spectral matching to interpret LC-MS/MS data during RNA modification mapping.

While a number of approaches have been developed to analyze liquid chromatography tandem mass spectrometry (LC-MS/MS) data obtained from modified oligonucleotides, the majority of these methods require analyzing every MS/MS spectrum de novo to sequence the oligonucleotide and place the modification. Spectral matching is an alternative approach for analyzing MS/MS data that is based on creating a library of annotated MS/MS spectra against which individual MS/MS data can be searched. Here, we have adapted the existing NIST spectral matching software to enable its use in the interpretation of MS/MS data obtained from modified oligonucleotides. In particular, we demonstrate the utility of this approach to identify specific post-transcriptionally modified nucleosides in particular transfer RNAs (tRNAs) obtained through a conventional RNA modification mapping experimental protocol. Spectral matching was found to be an efficient approach for screening for known modified tRNAs by using the experimental data as the library and previously annotated RNase T1 digestion products of tRNAs as the reference spectra. The utility of spectral matching for rapid analysis of multiple LC-MS/MS analyses was demonstrated by screening mutant strains of Streptococcus mutans to identify the enzyme(s) responsible for synthesizing the tRNA position 37 modification threonylcarbamoyladenosine (t6 A).

Mapping m6A and m1A with mutation signatures.

In two novel RNA modification mapping methods, the authors have engineered RNA enzymes and used the enzyme-mediated mutational signatures to map m6A and m1A at single-nucleotide resolution in mammalian RNA.

TRNA Modification Profiles and Codon-Decoding Strategies in Methanocaldococcus jannaschii.

tRNAs play a critical role in mRNA decoding, and posttranscriptional modifications within tRNAs drive decoding efficiency and accuracy. The types and positions of tRNA modifications in model bacteria have been extensively studied, and tRNA modifications in a few eukaryotic organisms have also been characterized and localized to particular tRNA sequences. However, far less is known regarding tRNA modifications in archaea. While the identities of modifications have been determined for multiple archaeal organisms, Haloferax volcanii is the only organism for which modifications have been extensively localized to specific tRNA sequences. To improve our understanding of archaeal tRNA modification patterns and codon-decoding strategies, we have used liquid chromatography and tandem mass spectrometry to characterize and then map posttranscriptional modifications on 34 of the 35 unique tRNA sequences of Methanocaldococcus jannaschii A new posttranscriptionally modified nucleoside, 5-cyanomethyl-2-thiouridine (cnm5s2U), was discovered and localized to position 34. Moreover, data consistent with wyosine pathway modifications were obtained beyond the canonical tRNAPhe as is typical for eukaryotes. The high-quality mapping of tRNA anticodon loops enriches our understanding of archaeal tRNA modification profiles and decoding strategies.IMPORTANCE While many posttranscriptional modifications in M. jannaschii tRNAs are also found in bacteria and eukaryotes, several that are unique to archaea were identified. By RNA modification mapping, the modification profiles of M. jannaschii tRNA anticodon loops were characterized, allowing a comparative analysis with H. volcanii modification profiles as well as a general comparison with bacterial and eukaryotic decoding strategies. This general comparison reveals that M. jannaschii, like H. volcanii, follows codon-decoding strategies similar to those used by bacteria, although position 37 appears to be modified to a greater extent than seen in H. volcanii.

EYE ON CHINA

CRISPR gene-editing not as precise as thought, warns of safety risks Insomnia spreads among young Chinese African swine fever virus isolated in Northeast China China launches new stem cell clinic programs Protein in human body could inhibit progress of HIV Chinese HEV vaccine begin clinical trial in U.S. New screening method for early diagnosis of liver cancer in hepatitis B carriers Merck collaborates with GenScript to accelerate cell and gene therapy industrialisation in China Shineco expands to capture industrial cannabis market Research from XJTLU advances RNA modification mapping

Methods for RNA Modification Mapping Using Deep Sequencing: Established and New Emerging Technologies.

New analytics of post-transcriptional RNA modifications have paved the way for a tremendous upswing of the biological and biomedical research in this field. This especially applies to methods that included RNA-Seq techniques, and which typically result in what is termed global scale modification mapping. In this process, positions inside a cell’s transcriptome are receiving a status of potential modification sites (so called modification calling), typically based on a score of some kind that issues from the particular method applied. The resulting data are thought to represent information that goes beyond what is contained in typical transcriptome data, and hence the field has taken to use the term “epitranscriptome”. Due to the high rate of newly published mapping techniques, a significant number of chemically distinct RNA modifications have become amenable to mapping, albeit with variegated accuracy and precision, depending on the nature of the technique. This review gives a brief overview of known techniques, and how they were applied to modification calling.

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.

Improving RNA modification mapping sequence coverage by LC-MS through a nonspecific RNase U2-E49A mutant

We report the identification and use of a mutant of the purine selective ribonuclease RNase U2 that randomly cleaves RNA in a manner that is directly compatible with RNA modification mapping by mass spectrometry. A number of RNase U2 mutants were generated using site-saturation mutagenesis. The enzyme activity and specificity were tested using oligonucleotide substrates, which revealed an RNase U2 E49A mutant with limited specificity and a tendency to undercut RNA. Using this mutant, RNA digestion conditions were optimized to yield long, overlapping digestion products, which improve sequence coverage in RNA modification mapping experiments. The analytical utility of this mutant was demonstrated by liquid chromatography tandem mass spectrometry (LC-MS/MS) mapping of several modified RNAs where 100% sequence coverage could be obtained using only a single enzymatic digestion. This new mutant facilitates more accurate and efficient RNA modification mapping than traditional highly base-specific RNases that are currently used.

Combining recombinant ribonuclease U2 and protein phosphatase for RNA modification mapping by liquid chromatography–mass spectrometry

Ribonuclease (RNase) mapping of modified nucleosides onto RNA sequences is limited by RNase availability. A codon-optimized gene for RNase U2, a purine selective RNase with preference for adenosine, has been designed for overexpression using Escherichia coli as the host. Optimal expression conditions were identified enabling generation of milligram-scale quantities of active RNase U2. RNase U2 digestion products were found to terminate in both 2′,3′-cyclic phosphates and 3′-linear phosphates. To generate a homogeneous 3′-linear phosphate set of products, an enzymatic approach was investigated. Bacteriophage lambda protein phosphatase was identified as the optimal enzyme for hydrolyzing cyclic phosphates from RNase U2 products. The compatibility of this enzymatic approach with liquid chromatography–tandem mass spectrometry (LC–MS/MS) RNA modification mapping was then demonstrated. RNase U2 digestion followed by subsequent phosphatase treatment generated nearly 100% 3′-phosphate-containing products that could be characterized by LC–MS/MS. In addition, bacteriophage lambda protein phosphatase can be used to introduce 18O labels within the 3′-phosphate of digestion products when incubated in the presence of H218O, allowing prior isotope labeling methods for mass spectrometry to include digestion products from RNase U2.

Transcriptome-Wide Mapping of 5-methylcytidine RNA Modifications in Bacteria, Archaea, and Yeast Reveals m5C within Archaeal mRNAs

The presence of 5-methylcytidine (m5C) in tRNA and rRNA molecules of a wide variety of organisms was first observed more than 40 years ago. However, detection of this modification was limited to specific, abundant, RNA species, due to the usage of low-throughput methods. To obtain a high resolution, systematic, and comprehensive transcriptome-wide overview of m5C across the three domains of life, we used bisulfite treatment on total RNA from both gram positive (B. subtilis) and gram negative (E. coli) bacteria, an archaeon (S. solfataricus) and a eukaryote (S. cerevisiae), followed by massively parallel sequencing. We were able to recover most previously documented m5C sites on rRNA in the four organisms, and identified several novel sites in yeast and archaeal rRNAs. Our analyses also allowed quantification of methylated m5C positions in 64 tRNAs in yeast and archaea, revealing stoichiometric differences between the methylation patterns of these organisms. Molecules of tRNAs in which m5C was absent were also discovered. Intriguingly, we detected m5C sites within archaeal mRNAs, and identified a consensus motif of AUCGANGU that directs methylation in S. solfataricus. Our results, which were validated using m5C-specific RNA immunoprecipitation, provide the first evidence for mRNA modifications in archaea, suggesting that this mode of post-transcriptional regulation extends beyond the eukaryotic domain.