Analysis of RNA modifications by liquid chromatography–tandem mass spectrometry

The analysis of RNA modifications is of high importance in order to address a wide range of biological questions. Therefore, a highly sensitive and accurate method such as liquid chromatography-tandem mass spectrometry (LC-MS/MS) has to be available. By using different LC-MS/MS procedures, it is not only possible to quantify very low amounts of RNA modifications, but also to detect probably unknown modified nucleosides. For these cases the dynamic multiple reaction monitoring and the neutral loss scan are the most common techniques. Here, we provide the whole workflow for analyzing RNA samples regarding their modification content. This includes an equipment list, the preparation of required…

LC-MS Analysis of Methylated RNA

The detection and quantification of methylated RNA can be beneficial to understand certain cellular regulation processes such as transcriptional modulation of gene expression, immune response, or epigenetic alterations. Therefore, it is necessary to have methods available, which are extremely sensitive and accurate, for instance liquid chromatography-tandem mass spectrometry (LC-MS/MS). Here, we describe the preparation of RNA samples by enzymatic hydrolysis and the subsequent analysis of ribonucleosides by LC-MS/MS via NLS (Neutral loss scan) and DMRM (Dynamic multiple reaction monitoring). Also, we provide variations of these methods including chromatographic techniques and different kind…

Bioconjugation of Small Molecules to RNA Impedes Its Recognition by Toll-Like Receptor 7

A fundamental mechanism of the innate immune system is the recognition, via extra- and intracellular pattern recognition receptors, of pathogen-associated molecular patterns. A prominent example is represented by foreign nucleic acids, triggering the activation of several signaling pathways. Among these, the endosomal toll-like receptor 7 (TLR7) is known to be activated by single stranded RNA (ssRNA), which can be specifically influenced through elements of sequence structure and posttranscriptional modifications. Furthermore, small molecules TLR7 agonists (smTLRa) are applied as boosting adjuvants in vaccination processes. In this context, covalent conjugations between adjuvant and vaccine…

Urmylation and tRNA thiolation functions of ubiquitin-like Uba4·Urm1 systems are conserved from yeast to man

AbstractThe ubiquitin-like protein Urm1 from budding yeast and its E1-like activator Uba4 have dual roles in protein urmylation and tRNA thiolation pathways. To study whether these are conserved among eukaryotes, we used gene shuffles to replace the yeast proteins by their human counterparts, hURM1 and hUBA4/MOCS3. As judged from biochemical and genetical assays, hURM1 and hUBA4 are functional in yeast, albeit at reduced efficiencies. They mediate urmylation of the peroxiredoxin Ahp1, a known urmylation target in yeast, and support tRNA thiolation. Similar to hUBA4, yeast Uba4 itself is modified by Urm1 and hURM1 suggesting target overlap between eukaryal urmylation pathways. In sum, our st…

Variable presence of 5-methylcytosine in commercial RNA and DNA

Nucleoside methylations and other nucleic acid modifications have recently encountered a surge in interest, prompted, among other things, by the detection of methylation and active demethylation of DNA and mRNA by similar mechanisms. In DNA, deoxycytidine methylation by Dnmt enzymes generates 5-methyldeoxycytidine,1 an important epigenetic mark that typically causes inactivation of transcription of the methylated promoter region. Recent exciting developments have shown that these marks are not concrete-cast, but can be actively removed by the oxidative action of TET enzymes,2 which generate, through a series of 2-electron oxidations, first hydroxymethylcytidine (hm5C), then formyldeoxycytid…

Dynamic modulation of Dnmt2-dependent tRNA methylation by the micronutrient queuine

Dnmt2 enzymes are cytosine-5 methyltransferases that methylate C38 of several tRNAs. We report here that the activities of two Dnmt2 homologs, Pmt1 from Schizosaccharomyces pombe and DnmA from Dictyostelium discoideum, are strongly stimulated by prior queuosine (Q) modification of the substrate tRNA. In vivo tRNA methylation levels were stimulated by growth of cells in queuine-containing medium; in vitro Pmt1 activity was enhanced on Q-containing RNA; and queuine-stimulated in vivo methylation was abrogated by the absence of the enzyme that inserts queuine into tRNA, eukaryotic tRNA-guanine transglycosylase. Global analysis of tRNA methylation in S. pombe showed a striking selectivity of Pm…

Next‐Generation Sequencing‐Based RiboMethSeq Protocol for Analysis of tRNA 2′‐O‐Methylation

Analysis of RNA modifications by traditional physico‐chemical approaches is labor  intensive,  requires  substantial  amounts  of  input  material  and  only  allows  site‐by‐site  measurements.  The  recent  development  of  qualitative  and  quantitative  approaches  based  on   next‐generation sequencing (NGS) opens new perspectives for the analysis of various cellular RNA  species.  The  Illumina  sequencing‐based  RiboMethSeq  protocol  was  initially  developed  and  successfully applied for mapping of ribosomal RNA (rRNA) 2′‐O‐methylations. This method also  gives excellent results in the quantitative analysis of rRNA modifications in different species and  under varying growth condi…

Absolute and relative quantification of RNA modifications via biosynthetic isotopomers

In the resurging field of RNA modifications, quantification is a bottleneck blocking many exciting avenues. With currently over 150 known nucleoside alterations, detection and quantification methods must encompass multiple modifications for a comprehensive profile. LC-MS/MS approaches offer a perspective for comprehensive parallel quantification of all the various modifications found in total RNA of a given organism. By feeding (13)C-glucose as sole carbon source, we have generated a stable isotope-labeled internal standard (SIL-IS) for bacterial RNA, which facilitates relative comparison of all modifications. While conventional SIL-IS approaches require the chemical synthesis of single mod…

Absolute Quantifizierung nicht‐kodierender RNA‐Spezies mittels Mikroskala‐Thermophorese

Absolute quantification of noncoding RNA by microscale thermophoresis

Abstract Accurate quantification of the copy numbers of noncoding RNA has recently emerged as an urgent problem, with impact on fields such as RNA modification research, tissue differentiation, and others. Herein, we present a hybridization‐based approach that uses microscale thermophoresis (MST) as a very fast and highly precise readout to quantify, for example, single tRNA species with a turnaround time of about one hour. We developed MST to quantify the effect of tRNA toxins and of heat stress and RNA modification on single tRNA species. A comparative analysis also revealed significant differences to RNA‐Seq‐based quantification approaches, strongly suggesting a bias due to tRNA modifica…

Variable presence of 5-methylcytosine in commercial RNA and DNA

5-methylcytosine (m5C, mC) is a naturally occurring nucleoside modification in both RNA and DNA. Its presence in DNA is a widely accepted epigenetic mark for transcription inactivation. In RNA, its appearance in different coding as well as non-coding RNA implies multiple functions, with regulation of gene expression as a common denominator. Here we report on the serendipitous discovery of m5C in synthetic oligonucleotides, which prompted a systematic quantification in synthetic DNA and RNA of academic as well as of commercial origin. For both types of oligonucleotides, m5C was identified by comparison of fragmentation pattern and retention time with authentic standards by highly sensitive L…