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…

Die stark wachsende chemische Vielfalt der RNA-Modifikationen enthält eine Thioacetalstruktur

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…

Recognition of Specified RNA Modifications by the Innate Immune System

Microbial nucleic acids have been described as important activators of human innate immune responses by triggering so-called pattern recognition receptors (PRRs) that are expressed on innate immune cells, including plasmacytoid dendritic cells and monocytes. Although host and microbial nucleic acids share pronounced chemical and structural similarities, they significantly differ in their posttranscriptional modification profile, allowing the host to discriminate between self and nonself. In this regard, ribose 2'-O-methylation has been discovered as suppressor of RNA-induced PRR activation. Although 2'-O-methylation occurs with higher frequencies in eukaryotic than in prokaryotic RNA, the i…

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…

Inside Cover: A Vastly Increased Chemical Variety of RNA Modifications Containing a Thioacetal Structure (Angew. Chem. Int. Ed. 26/2018)

Double methylation of tRNA-U54 to 2′-O-methylthymidine (Tm) synergistically decreases immune response by Toll-like receptor 7

Abstract Sensing of nucleic acids for molecular discrimination between self and non-self is a challenging task for the innate immune system. RNA acts as a potent stimulus for pattern recognition receptors including in particular human Toll-like receptor 7 (TLR7). Certain RNA modifications limit potentially harmful self-recognition of endogenous RNA. Previous studies had identified the 2′-O-methylation of guanosine 18 (Gm18) within tRNAs as an antagonist of TLR7 leading to an impaired immune response. However, human tRNALys3 was non-stimulatory despite lacking Gm18. To identify the underlying molecular principle, interferon responses of human peripheral blood mononuclear cells to differentia…

Innentitelbild: Die stark wachsende chemische Vielfalt der RNA-Modifikationen enthält eine Thioacetalstruktur (Angew. Chem. 26/2018)

A Vastly Increased Chemical Variety of RNA Modifications Containing a Thioacetal Structure

International audience; Recently discovered new chemical entities in RNA modifications have involved surprising functional groups that enlarge the chemical space of RNA. Using LC-MS, we found over 100 signals of RNA constituents that contained a ribose moiety in tRNAs from E. coli. Feeding experiments with variegated stable isotope labeled compounds identified 37 compounds that are new structures of RNA modifications. One structure was elucidated by deuterium exchange and high-resolution mass spectrometry. The structure of msms2 i6 A (2-methylthiomethylenethio-N6-isopentenyl-adenosine) was confirmed by methione-D3 feeding experiments and by synthesis of the nucleobase. The msms2 i6 A contai…

A protein-RNA interaction atlas of the ribosome biogenesis factor AATF

AbstractAATF is a central regulator of the cellular outcome upon p53 activation, a finding that has primarily been attributed to its function as a transcription factor. Recent data showed that AATF is essential for ribosome biogenesis and plays a role in rRNA maturation. AATF has been implicated to fulfil this role through direct interaction with rRNA and was identified in several RNA-interactome capture experiments. Here, we provide a first comprehensive analysis of the RNA bound by AATF using CLIP-sequencing. Interestingly, this approach shows predominant binding of the 45S pre-ribosomal RNA precursor molecules. Furthermore, AATF binds to mRNAs encoding for ribosome biogenesis factors as …

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…