Machine learning of reverse transcription signatures of variegated polymerases allows mapping and discrimination of methylated purines in limited transcriptomes

AbstractReverse transcription (RT) of RNA templates containing RNA modifications leads to synthesis of cDNA containing information on the modification in the form of misincorporation, arrest, or nucleotide skipping events. A compilation of such events from multiple cDNAs represents an RT-signature that is typical for a given modification, but, as we show here, depends also on the reverse transcriptase enzyme. A comparison of 13 different enzymes revealed a range of RT-signatures, with individual enzymes exhibiting average arrest rates between 20 and 75%, as well as average misincorporation rates between 30 and 75% in the read-through cDNA. Using RT-signatures from individual enzymes to trai…

2'-O-methylation within prokaryotic and eukaryotic tRNA inhibits innate immune activation by endosomal Toll-like receptors but does not affect recognition of whole organisms

Bacterial RNA has emerged as an important activator of innate immune responses by stimulating Toll-like receptors TLR7 and TLR8 in humans. Guanosine 2′-O-methylation at position 18 (Gm18) in bacterial tRNA was shown to antagonize tRNA-induced TLR7/8 activation, suggesting a potential role of Gm18 as an immune escape mechanism. This modification also occurs in eukaryotic tRNA, yet a physiological immune function remained to be tested. We therefore set out to investigate the immune modulatory role of Gm18 in both prokaryotic and eukaryotic microorganisms, Escherichia coli and Saccharomyces cerevisiae, and in human cells. Using RiboMethSeq analysis we show that mutation of trmH in E. coli, trm…

High-Throughput Mapping of 2′-O-Me Residues in RNA Using Next-Generation Sequencing (Illumina RiboMethSeq Protocol)

Detection of RNA modifications in native RNAs is a tedious and laborious task, since the global level of these residues is low and most of the suitable physico-chemical methods require purification of the RNA of interest almost to homogeneity. To overcome these limitations, methods based on RT-driven primer extension have been developed and successfully used, sometimes in combination with a specific chemical treatment. Nowadays, some of these approaches have been coupled to high-throughput sequencing technologies, allowing the access to transcriptome-wide data. RNA 2'-O-methylation is one of the ubiquitous nucleotide modifications found in many RNA types from bacteria, archaea, and eukarya.…

Non-Redundant tRNA Reference Sequences for Deep Sequencing Analysis of tRNA Abundance and Epitranscriptomic RNA Modifications

Analysis of RNA by deep-sequencing approaches has found widespread application in modern biology. In addition to measurements of RNA abundance under various physiological conditions, such techniques are now widely used for mapping and quantification of RNA modifications. Transfer RNA (tRNA) molecules are among the frequent targets of such investigation, since they contain multiple modified residues. However, the major challenge in tRNA examination is related to a large number of duplicated and point-mutated genes encoding those RNA molecules. Moreover, the existence of multiple isoacceptors/isodecoders complicates both the analysis and read mapping. Existing databases for tRNA sequencing pr…

AlkAniline-Seq: A Highly Sensitive and Specific Method for Simultaneous Mapping of 7-Methyl-guanosine (m7G) and 3-Methyl-cytosine (m3C) in RNAs by High-Throughput Sequencing

Epitranscriptomics is an emerging field where the development of high-throughput analytical technologies is essential to profile the dynamics of RNA modifications under different conditions. Despite important advances during the last 10 years, the number of RNA modifications detectable by next-generation sequencing is restricted to a very limited subset. Here, we describe a highly efficient and fast method called AlkAniline-Seq to map simultaneously two different RNA modifications: 7-methyl-guanosine (m7G) and 3-methyl-cytosine (m3C) in RNA. Our protocol is based on three subsequent chemical/enzymatic steps allowing the enrichment of RNA fragments ending at position n + 1 to the modified nu…

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

Use of Specific Chemical Reagents for Detection of Modified Nucleotides in RNA

Naturally occurring cellular RNAs contain an impressive number of chemically distinct modified residues which appear posttranscriptionally, as a result of specific action of the corresponding RNA modification enzymes. Over 100 different chemical modifications have been identified and characterized up to now. Identification of the chemical nature and exact position of these modifications is typically based on 2D-TLC analysis of nucleotide digests, on HPLC coupled with mass spectrometry, or on the use of primer extension by reverse transcriptase. However, many modified nucleotides are silent in reverse transcription, since the presence of additional chemical groups frequently does not change …

RNA nucleotide methylation

Methylation of RNA occurs at a variety of atoms, nucleotides, sequences and tertiary structures. Strongly related to other posttranscriptional modifications, methylation of different RNA species includes tRNA, rRNA, mRNA, tmRNA, snRNA, snoRNA, miRNA, and viral RNA. Different catalytic strategies are employed for RNA methylation by a variety of RNA-methyltransferases which fall into four superfamilies. This review outlines the different functions of methyl groups in RNA, including biophysical, biochemical and metabolic stabilization of RNA, quality control, resistance to antibiotics, mRNA reading frame maintenance, deciphering of normal and altered genetic code, selenocysteine incorporation,…

Expanding the chemical scope of RNA:methyltransferases to site-specific alkynylation of RNA for click labeling.

This work identifies the combination of enzymatic transfer and click labeling as an efficient method for the site-specific tagging of RNA molecules for biophysical studies. A double-activated analog of the ubiquitous co-substrate S-adenosyl-l-methionine was employed to enzymatically transfer a five carbon chain containing a terminal alkynyl moiety onto RNA. The tRNA:methyltransferase Trm1 transferred the extended alkynyl moiety to its natural target, the N2 of guanosine 26 in tRNA(Phe). LC/MS and LC/MS/MS techniques were used to detect and characterize the modified nucleoside as well as its cycloaddition product with a fluorescent azide. The latter resulted from a labeling reaction via Cu(I…

Engineering of a DNA Polymerase for Direct m6A Sequencing

Methods for the detection of RNA modifications are of fundamental importance for advancing epitranscriptomics. N6-methyladenosine (m6A) is the most abundant RNA modification in mammalian mRNA and is involved in the regulation of gene expression. Current detection techniques are laborious and rely on antibody-based enrichment of m6A-containing RNA prior to sequencing, since m6A modifications are generally "erased" during reverse transcription (RT). To overcome the drawbacks associated with indirect detection, we aimed to generate novel DNA polymerase variants for direct m6A sequencing. Therefore, we developed a screen to evolve an RT-active KlenTaq DNA polymerase variant that sets a mark for…

Pseudouridine: Still mysterious, but never a fake (uridine)!

International audience; Pseudouridine () is the most abundant of >150 nucleoside modifications in RNA. Although was discovered as the first modified nucleoside more than half a century ago, neither the enzymatic mechanism of its formation, nor the function of this modification are fully elucidated. We present the consistent picture of synthases, their substrates and their substrate positions in model organisms of all domains of life as it has emerged to date and point out the challenges that remain concerning higher eukaryotes and the elucidation of the enzymatic mechanism.

CoverageAnalyzer (CAn): A Tool for Inspection of Modification Signatures in RNA Sequencing Profiles

Combination of reverse transcription (RT) and deep sequencing has emerged as a powerful instrument for the detection of RNA modifications, a field that has seen a recent surge in activity because of its importance in gene regulation. Recent studies yielded high-resolution RT signatures of modified ribonucleotides relying on both sequence-dependent mismatch patterns and reverse transcription arrests. Common alignment viewers lack specialized functionality, such as filtering, tailored visualization, image export and differential analysis. Consequently, the community will profit from a platform seamlessly connecting detailed visual inspection of RT signatures and automated screening for modifi…

Graphical Workflow System for Modification Calling by Machine Learning of Reverse Transcription Signatures

Modification mapping from cDNA data has become a tremendously important approach in epitranscriptomics. So-called reverse transcription signatures in cDNA contain information on the position and nature of their causative RNA modifications. Data mining of, e.g. Illumina-based high-throughput sequencing data, is therefore fast growing in importance, and the field is still lacking effective tools. Here we present a versatile user-friendly graphical workflow system for modification calling based on machine learning. The workflow commences with a principal module for trimming, mapping, and postprocessing. The latter includes a quantification of mismatch and arrest rates with single-nucleotide re…

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

NOseq: amplicon sequencing evaluation method for RNA m6A sites after chemical deamination

Abstract Methods for the detection of m6A by RNA-Seq technologies are increasingly sought after. We here present NOseq, a method to detect m6A residues in defined amplicons by virtue of their resistance to chemical deamination, effected by nitrous acid. Partial deamination in NOseq affects all exocyclic amino groups present in nucleobases and thus also changes sequence information. The method uses a mapping algorithm specifically adapted to the sequence degeneration caused by deamination events. Thus, m6A sites with partial modification levels of ∼50% were detected in defined amplicons, and this threshold can be lowered to ∼10% by combination with m6A immunoprecipitation. NOseq faithfully d…

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…

The ribose methylation enzyme FTSJ1 has a conserved role in neuron morphology and learning performance

ABSTRACTFTSJ1 is a conserved human 2’-O-methyltransferase (Nm-MTase) that modifies several transfer RNAs (tRNAs) at position 32 and the wobble position 34 in the AntiCodon Loop (ACL). Its loss of function has been linked to Non-Syndromic X-Linked Intellectual Disability (NSXLID), and more recently to cancers. However, the molecular mechanisms underlying these pathologies are currently unclear. Here we report a novelFTSJ1pathogenic variant from a NSXLID patient. Using blood cells derived from this patient and other affected individuals carryingFTSJ1mutations, we performed an unbiased and comprehensive RiboMethSeq analysis to map the ribose methylation (Nm) on all human tRNAs and identify nov…

Positioning Europe for the EPITRANSCRIPTOMICS challenge

WOS: 000444092300018 PubMed ID: 29671387 The genetic alphabet consists of the four letters: C, A, G, and T in DNA and C,A,G, and U in RNA. Triplets of these four letters jointly encode 20 different amino acids out of which proteins of all organisms are built. This system is universal and is found in all kingdoms of life. However, bases in DNA and RNA can be chemically modified. In DNA, around 10 different modifications are known, and those have been studied intensively over the past 20years. Scientific studies on DNA modifications and proteins that recognize them gave rise to the large field of epigenetic and epigenomic research. The outcome of this intense research field is the discovery t…

Eukaryotic rRNA Modification by Yeast 5-Methylcytosine-Methyltransferases and Human Proliferation-Associated Antigen p120.

International audience; Modified nucleotide 5-methylcytosine (m(5)C) is frequently present in various eukaryotic RNAs, including tRNAs, rRNAs and in other non-coding RNAs, as well as in mRNAs. RNA: m(5)C-methyltranferases (MTases) Nop2 from S. cerevisiae and human proliferation-associated nucleolar antigen p120 are both members of a protein family called Nop2/NSUN/NOL1. Protein p120 is well-known as a tumor marker which is over-expressed in various cancer tissues. Using a combination of RNA bisulfite sequencing and HPLC-MS/MS analysis, we demonstrated here that p120 displays an RNA:m(5)C-MTase activity, which restores m(5)C formation at position 2870 in domain V of 25S rRNA in a nop2 Delta …

Analysis of pseudouridines and other RNA modifications using hydraPsiSeq protocol

Detection of RNA modified nucleotides using deep sequencing can be performed by several approaches, including antibody-driven enrichment and natural or chemically induced RT signatures. However, only very few RNA modified nucleotides generate natural RT signatures and antibody-driven enrichment heavily depends on the quality of antibodies used and may be highly biased. Thus, the use of chemically-induced RT signatures is now considered as the most trusted experimental approach. In addition, the use of chemical reagents allows inclusion of simple "mock-treated" controls, to exclude spontaneous RT arrests, SNPs and other misincorporation-prone sites. Hydrazine is a well-known RNA-specific rea…

DNA and RNA Pyrimidine Nucleobase Alkylation at the Carbon-5 Position

International audience; The carbon 5 of pyrimidine nucleobases is a privileged position in terms of nucleoside modification in both DNA and RNA. The simplest modification of uridine at this position is methylation leading to thymine. Thymine is an integral part of the standard nucleobase repertoire of DNA that is synthesized at the nucleotide level. However, it also occurs in RNA, where it is synthesized posttranscriptionally at the polynucleotide level. The cytidine analogue 5-methylcytidine also occurs in both DNA and RNA, but is introduced at the polynucleotide level in both cases. The same applies to a plethora of additional derivatives found in nature, resulting either from a direct mo…

Manganese Ions Individually Alter the Reverse Transcription Signature of Modified Ribonucleosides

Reverse transcription of RNA templates containing modified ribonucleosides transfers modification-related information as misincorporations, arrest or nucleotide skipping events to the newly synthesized cDNA strand. The frequency and proportion of these events, merged from all sequenced cDNAs, yield a so-called RT signature, characteristic for the respective RNA modification and reverse transcriptase (RT). While known for DNA polymerases in so-called error-prone PCR, testing of four different RTs by replacing Mg2+ with Mn2+ in reaction buffer revealed the immense influence of manganese chloride on derived RT signatures, with arrest rates on m1A positions dropping from 82% down to 24%. Additi…

High-throughput sequencing for 1-methyladenosine (m1A) mapping in RNA

Abstract Detection and mapping of modified nucleotides in RNAs is a difficult and laborious task. Several physico-chemical approaches based on differential properties of modified nucleotides can be used, however, most of these methods do not allow high-throughput analysis. Here we describe in details a method for mapping of rather common 1-methyladenosine (m1A) residues using high-throughput next generation sequencing (NGS). Since m1A residues block primer extension during reverse transcription (RT), the accumulation of abortive products as well as the nucleotide misincorporation can be detected in the sequencing data. The described library preparation protocol allows to capture both types …

Detecting RNA modifications in the epitranscriptome: predict and validate

RNA modifications are emerging players in the field of post-transcriptional regulation of gene expression, and are attracting a comparable degree of research interest to DNA and histone modifications in the field of epigenetics. We now know of more than 150 RNA modifications and the true potential of a few of these is currently emerging as the consequence of a leap in detection technology, principally associated with high-throughput sequencing. This Review outlines the major developments in this field through a structured discussion of detection principles, lays out advantages and drawbacks of new high-throughput methods and presents conventional biophysical identification of modifications …

tRNA stabilization by modified nucleotides.

Post-transcriptional ribonucleotide modification is a phenomenon best studied in tRNA, where it occurs most frequently and in great chemical diversity. This paper reviews the intrinsic network of modifications in the structural core of the tRNA, which governs structural flexibility and rigidity to fine-tune the molecule to peak performance and to regulate its steady-state level. Structural effects of RNA modifications range from nanometer-scale rearrangements to subtle restrictions of conformational space on the angstrom scale. Structural stabilization resulting from nucleotide modification results in increased thermal stability and translates into protection against unspecific degradation …

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…

Balancing of mitochondrial translation through METTL8-mediated m3C modification of mitochondrial tRNAs.

Mitochondria contain a specific translation machinery for the synthesis of mitochondria-encoded respiratory chain components. Mitochondrial tRNAs (mt-tRNAs) are also generated from the mitochondrial DNA and, similar to their cytoplasmic counterparts, are post-transcriptionally modified. Here, we find that the RNA methyltransferase METTL8 is a mitochondrial protein that facilitates 3-methyl-cytidine (m3C) methylation at position C32 of the mt-tRNASer(UCN) and mt-tRNAThr. METTL8 knockout cells show a reduction in respiratory chain activity, whereas overexpression increases activity. In pancreatic cancer, METTL8 levels are high, which correlates with lower patient survival and an enhanced resp…

Illumina-based RiboMethSeq approach for mapping of 2'-O-Me residues in RNA

International audience; RNA 2'-O-methylation is one of the ubiquitous nucleotide modifications found in many RNA types from Bacteria, Archaea and Eukarya. RNAs bearing 2'-O-methylations show increased resistance to degradation and enhanced stability in helices. While the exact role of each 2'-O-Me residue remained elusive, the catalytic protein Fibrillarin (Nop1 in yeast) responsible for 2'-O-methylation in eukaryotes, is associated with human pathologies. Therefore, there is an urgent need to precisely map and quantify hundreds of 2'-O-Me residues in RNA using high-throughput technologies. Here, we develop a reliable protocol using alkaline fragmentation of total RNA coupled to a commonly …

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 …

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

A multifunctional bioconjugate module for versatile photoaffinity labeling and click chemistry of RNA

A multifunctional reagent based on a coumarin scaffold was developed for derivatization of naive RNA. The alkylating agent N3BC [7-azido-4-(bromomethyl)coumarin], obtained by Pechmann condensation, is selective for uridine. N3BC and its RNA conjugates are pre-fluorophores which permits controlled modular and stepwise RNA derivatization. The success of RNA alkylation by N3BC can be monitored by photolysis of the azido moiety, which generates a coumarin fluorophore that can be excited with UV light of 320 nm. The azidocoumarin-modified RNA can be flexibly employed in structure-function studies. Versatile applications include direct use in photo-crosslinking studies to cognate proteins, as dem…

AlkAniline-Seq: Profiling of m7 G and m3 C RNA Modifications at Single Nucleotide Resolution.

RNA modifications play essential roles in gene expression regulation. Only seven out of >150 known RNA modifications are detectable transcriptome-wide by deep sequencing. Here we describe a new principle of RNAseq library preparation, which relies on a chemistry based positive enrichment of reads in the resulting libraries, and therefore leads to unprecedented signal-to-noise ratios. The proposed approach eschews conventional RNA sequencing chemistry and rather exploits the generation of abasic sites and subsequent aniline cleavage. The newly generated 5'-phosphates are used as unique entry for ligation of an adapter in library preparation. This positive selection, embodied in the AlkAnilin…

The reverse transcription signature of N-1-methyladenosine in RNA-Seq is sequence dependent

The combination of Reverse Transcription (RT) and high-throughput sequencing has emerged as a powerful combination to detect modified nucleotides in RNA via analysis of either abortive RT-products or of the incorporation of mismatched dNTPs into cDNA. Here we simultaneously analyze both parameters in detail with respect to the occurrence of N-1-methyladenosine (m1A) in the template RNA. This naturally occurring modification is associated with structural effects, but it is also known as a mediator of antibiotic resistance in ribosomal RNA. In structural probing experiments with dimethylsulfate, m1A is routinely detected by RT-arrest. A specifically developed RNA-Seq protocol was tailored to …

Mapping of 7-methylguanosine (m7G), 3-methylcytidine (m3C), dihydrouridine (D) and 5-hydroxycytidine (ho5C) RNA modifications by AlkAniline-Seq

Precise and reliable mapping of modified nucleotides in RNA is a challenging task in epitranscriptomics analysis. Only deep sequencing-based methods are able to provide both, a single-nucleotide resolution and sufficient selectivity and sensitivity. A number of protocols employing specific chemical reagents to distinguish modified RNA nucleotides from canonical parental residues have already proven their performance. We developed a deep-sequencing analytical pipeline for simultaneous detection of several modified nucleotides of different nature (methylation, hydroxylation, reduction) in RNA. The AlkAniline-Seq protocol uses intrinsic fragility of the N-glycosidic bond present in certain mod…

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…

Holistic Optimization of Bioinformatic Analysis Pipeline for Detection and Quantification of 2′-O-Methylations in RNA by RiboMethSeq

International audience; A major trend in the epitranscriptomics field over the last 5 years has been the high-throughput analysis of RNA modifications by a combination of specific chemical treatment(s), followed by library preparation and deep sequencing. Multiple protocols have been described for several important RNA modifications, such as 5-methylcytosine (m5C), pseudouridine (ψ), 1-methyladenosine (m1A), and 2'-O-methylation (Nm). One commonly used method is the alkaline cleavage-based RiboMethSeq protocol, where positions of reads' 5'-ends are used to distinguish nucleotides protected by ribose methylation. This method was successfully applied to detect and quantify Nm residues in vari…

Use of Specific Chemical Reagents for Detection of Modified Nucleotides in RNA

International audience; Naturally occurring cellular RNAs contain an impressive number of chemically distinct modified residues which appear posttranscriptionally, as a result of specific action of the corresponding RNA modification enzymes. Over 100 different chemical modifications have been identified and characterized up to now. Identification of the chemical nature and exact position of these modifications is typically based on 2D-TLC analysis of nucleotide digests, on HPLC coupled with mass spectrometry, or on the use of primer extension by reverse transcriptase. However, many modified nucleotides are silent in reverse transcription, since the presence of additional chemical groups fre…

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

Mapping and Quantification of tRNA 2′-O-Methylation by RiboMethSeq

Current development of epitranscriptomics field requires efficient experimental protocols for precise mapping and quantification of various modified nucleotides in RNA. Despite important advances in the field during the last 10 years, this task is still extremely laborious and time-consuming, even when high-throughput analytical approaches are employed. Moreover, only a very limited subset of RNA modifications can be detected and only rarely be quantified by these powerful techniques. In the past, we developed and successfully applied alkaline fragmentation-based RiboMethSeq approach for mapping and precise quantification of multiple 2'-O-methylation residues in ribosomal RNA. Here we descr…

Entwicklung einer DNA-Polymerase für die direkte m6A-Sequenzierung

Methoden zur Analyse von RNA-Modifikationen sind essenziell fur das Forschungsfeld der Epitranskriptomik. N6-Methyladenosin (m6A) ist die haufigste Modifikation in der mRNA von Saugetieren und erfullt Funktionen in der Regulation der Genexpression. Techniken zur Detektion dieser Modifikation basieren derzeit auf der Anreicherung von m6A-haltigen RNA-Fragmenten durch Antikorper. Dieser m6A-spezifische Schritt vor der Sequenzierung ist notig, da die Information uber die Modifikation wahrend der reversen Transkription (RT) geloscht wird. Um die Nachteile einer solchen indirekten Detektion zu uberwinden, haben wir uns zum Ziel gesetzt, neue DNA-Polymerasen zu entwickeln, die eine direkte m6A-Se…

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…

General Principles for the Detection of Modified Nucleotides in RNA by Specific Reagents.

Epitranscriptomics heavily rely on chemical reagents for the detection, quantification, and localization of modified nucleotides in transcriptomes. Recent years have seen a surge in mapping methods that use innovative and rediscovered organic chemistry in high throughput approaches. While this has brought about a leap of progress in this young field, it has also become clear that the different chemistries feature variegated specificity and selectivity. The associated error rates, e.g., in terms of false positives and false negatives, are in large part inherent to the chemistry employed. This means that even assuming technically perfect execution, the interpretation of mapping results issuin…

Limited antibody specificity compromises epitranscriptomic analyses

International audience; A controversial discussion on the occurrence of the RNA modification m1A in mRNA takes a new turn, as an antibody with a central role in modification mapping was shown to also bind mRNA cap structures.