Selective terminal methylation of a tRNA wobble base.

Active tRNAs are extensively post-transcriptionally modified, particularly at the wobble position 34 and the position 37 on the 3'-side of the anticodon. The 5-carboxy-methoxy modification of U34 (cmo5U34) is present in Gram-negative tRNAs for six amino acids (Ala, Ser, Pro, Thr, Leu and Val), four of which (Ala, Ser, Pro and Thr) have a terminal methyl group to form 5-methoxy-carbonyl-methoxy-uridine (mcmo5U34) for higher reading-frame accuracy. The molecular basis for the selective terminal methylation is not understood. Many cmo5U34-tRNAs are essential for growth and cannot be substituted for mutational analysis. We show here that, with a novel genetic approach, we have created and isolated mutants of Escherichia coli tRNAPro and tRNAVal for analysis of the selective terminal methylation. We show that substitution of G35 in the anticodon of tRNAPro inactivates the terminal methylation, whereas introduction of G35 to tRNAVal confers it, indicating that G35 is a major determinant for the selectivity. We also show that, in tRNAPro, the terminal methylation at U34 is dependent on the primary m1G methylation at position 37 but not vice versa, indicating a hierarchical ranking of modifications between positions 34 and 37. We suggest that this hierarchy provides a mechanism to ensure top performance of a tRNA inside of cells.

The t6A modification acts as a positive determinant for the anticodon nuclease PrrC, and is distinctively nonessential in Streptococcus mutans.

Endoribonuclease toxins (ribotoxins) are produced by bacteria and fungi to respond to stress, eliminate non-self competitor species, or interdict virus infection. PrrC is a bacterial ribotoxin that targets and cleaves tRNALysUUU in the anticodon loop. In vitro studies suggested that the post-transcriptional modification threonylcarbamoyl adenosine (t6A) is required for PrrC activity but this prediction had never been validated in vivo. Here, by using t6A-deficient yeast derivatives, it is shown that t6A is a positive determinant for PrrC proteins from various bacterial species. Streptococcus mutans is one of the few bacteria where the t6A synthesis gene tsaE (brpB) is dispensable and its genome encodes a PrrC toxin. We had previously shown using an HPLC-based assay that the S. mutans tsaE mutant was devoid of t6A. However, we describe here a novel and a more sensitive hybridization-based t6A detection method (compared to HPLC) that showed t6A was still present in the S. mutans ΔtsaE, albeit at greatly reduced levels (93% reduced compared with WT). Moreover, mutants in 2 other S. mutans t6A synthesis genes (tsaB and tsaC) were shown to be totally devoid of the modification thus confirming its dispensability in this organism. Furthermore, analysis of t6A modification ratios and of t6A synthesis genes mRNA levels in S. mutans suggest they may be regulated by growth phase.

Evaluating orthogonality between ion-pair reversed phase, anion exchange, and hydrophilic interaction liquid chromatography for the separation of synthetic oligonucleotides.

The orthogonality of separation between ion-pair reversed phase (IP-RP), anion exchange (AEX), and hydrophilic interaction liquid chromatography (HILIC) was evaluated for oligonucleotides. A polythymidine standard ladder was first used to evaluate the three methods and showed zero orthogonality, where retention and selectivity were based on oligonucleotide charge/size under all three conditions. Next, a model 23-mer synthetic oligonucleotide containing 4 phosphorothioate bonds with 2' fluoro and 2'-O-methyl ribose modifications typical of small interfering RNA was used for evaluating orthogonality. The resolution and orthogonality were evaluated between the three modes of chromatography in terms of selectivity differences for nine common impurities, including truncations (n-1, n-2), addition (n + 1), oxidation, and de-fluorination. We first evaluated different ion-pairing reagents that provided the best separation of the key impurities while suppressing diastereomer separation due to phosphorothioate linkages. Although different ion-pairing reagents affected resolution, very little orthogonality was observed. We then compared the retention times between IP-RP, HILIC, and AEX for each impurity of the model oligonucleotide and observed various selectivity changes. The results suggest that coupling HILIC with either AEX or IP-RP provide the highest degree of orthogonality due to the differences in retention for hydrophilic nucleobases and modifications under HILIC conditions. IP-RP provided the highest overall resolution for the impurity mixture, whereas more co-elution was observed with HILIC and AEX. The unique selectivity patterns offered by HILIC provides an interesting alternative to IP-RP or AEX, in addition to the potential for coupling with multidimensional separations. Future work should explore orthogonality for oligonucleotides with subtle sequence differences such as nucleobase modifications and base flip isomers, longer strands such as guide RNA and messenger RNA, and other biotherapeutic modalities such as peptides, antibodies, and antibody-drug-conjugates.

Ionizing radiation and chemical oxidant exposure impacts on Cryptococcus neoformans transfer RNAs.

Cryptococcus neoformans is a fungus that is able to survive abnormally high levels of ionizing radiation (IR). The radiolysis of water by IR generates reactive oxygen species (ROS) such as H2O2 and OH-. C. neoformans withstands the damage caused by IR and ROS through antioxidant production and enzyme-catalyzed breakdown of ROS. Given these particular cellular protein needs, questions arise whether transfer ribonucleic acids molecules (tRNAs) undergo unique chemical modifications to maintain their structure, stability, and/or function under such environmental conditions. Here, we investigated the effects of IR and H2O2 exposure on tRNAs in C. neoformans. We experimentally identified the modified nucleosides present in C. neoformans tRNAs and quantified changes in those modifications upon exposure to oxidative conditions. To better understand these modified nucleoside results, we also evaluated tRNA pool composition in response to the oxidative conditions. We found that regardless of environmental conditions, tRNA modifications and transcripts were minimally affected. A rationale for the stability of the tRNA pool and its concomitant profile of modified nucleosides is proposed based on the lack of codon bias throughout the C. neoformans genome and in particular for oxidative response transcripts. Our findings suggest that C. neoformans can rapidly adapt to oxidative environments as mRNA translation/protein synthesis are minimally impacted by codon bias.

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).

Stable Isotope Labeling for Improved Comparative Analysis of RNA Digests by Mass Spectrometry.

Even with the advent of high throughput methods to detect modified ribonucleic acids (RNAs), mass spectrometry remains a reliable method to detect, characterize, and place post-transcriptional modifications within an RNA sequence. Here we have developed a stable isotope labeling comparative analysis of RNA digests (SIL-CARD) approach, which improves upon the original 18O/16O labeling CARD method. Like the original, SIL-CARD allows sequence or modification information from a previously uncharacterized in vivo RNA sample to be obtained by direct comparison with a reference RNA, the sequence of which is known. This reference is in vitro transcribed using a 13C/15N isotopically enriched nucleoside triphosphate (NTP). The two RNAs are digested with an endonuclease, the specificity of which matches the labeled NTP used for transcription. As proof of concept, several transfer RNAs (tRNAs) were characterized by SIL-CARD, where labeled guanosine triphosphate was used for the reference in vitro transcription. RNase T1 digestion products from the in vitro transcript will be 15 Da higher in mass than the same digestion products from the in vivo tRNA that are unmodified, leading to a doublet in the mass spectrum. Singlets, rather than doublets, arise if a sequence variation or a post-transcriptional modification is present that results in a relative mass shift different from 15 Da. Moreover, the use of the in vitro synthesized tRNA transcript allows for quantitative measurement of RNA abundance. Overall, SIL-CARD simplifies data analysis and enhances quantitative RNA modification mapping by mass spectrometry. Graphical Abstract ᅟ.

Going global: the new era of mapping modifications in RNA.

The post-transcriptional modification of RNA by the addition of one or more chemical groups has been known for over 50 years. These chemical modifications, once thought to be static, are now being discovered to play key regulatory roles in gene expression. The advent of massive parallel sequencing of RNA (RNA-seq) now allows us to probe the complexity of cellular RNA and how chemically altering RNA structure expands the RNA vocabulary. Here we present an overview of the various strategies and technologies that are available to profile RNA chemical modifications at the cellular level. These strategies can be characterized as targeted and untargeted approaches: targeted strategies are developed for one single chemical modification while untargeted strategies are more broadly applicable to a range of such chemical changes. Key for all of these approaches is the ability to locate modifications within the RNA sequence. While most of these methods are built upon an RNA-Seq pipeline, alternative approaches based on mass spectrometry or conventional DNA sequencing retain value in the overall analysis process. We also look forward toward future opportunities and technologies that may expand the types of modifications that can be globally profiled. Given the ever increasing recognition that these RNA chemical modifications play important biological roles, a variety of methods, preferably orthogonal approaches, will be required to globally identify, validate and quantify RNA chemical modifications found in the transcriptome. WIREs RNA 2017, 8:e1367. doi: 10.1002/wrna.1367 For further resources related to this article, please visit the WIREs website.

Comparative Analysis of Ribonucleic Acid Digests (CARD) by Mass Spectrometry.

We describe the comparative analysis of ribonucleic acid digests (CARD) approach for RNA modification analysis. This approach employs isotope labeling during RNase digestion, which allows the direct comparison of a tRNA of unknown modification status against a reference tRNA, whose sequence or modification status is known. The reference sample is labeled with 18O during RNase digestion while the candidate (unknown) sample is labeled with 16O. These RNase digestion products are combined and analyzed by mass spectrometry. Identical RNase digestion products will appear in the mass spectrum as characteristic doublets, separated by 2 Da due to the 16O/18O mass difference. Singlets arise in the mass spectrum when the sequence or modification status of a particular RNase digestion product from the reference is not matched in the candidate (unknown) sample. This CARD approach for RNA modification analysis simplifies the determination of differences between reference and candidate samples, providing a route for higher throughput screening of samples for modification profiles, including determination of tRNA methylation patterns.