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  • Review Article
  • Published:

Detecting RNA modifications in the epitranscriptome: predict and validate

Nature Reviews Genetics volume 18, pages 275–291 (2017)Cite this article

Subjects

Key Points

  • Modified nucleotides are present in many RNA species and have multiple roles in the global regulation and fine-tuning of gene expression.

  • Despite growing interest in the discovery and analysis of RNA modification profiles and their dynamics, conventional methods for transcriptome-wide profiling are laborious and time-consuming.

  • Next-generation sequencing brings new perspectives for global analysis of RNA modification in different cells and tissues under various physiological conditions. Current methods and technologies applied for RNA modification analysis at the transcriptome level are discussed.

  • Most of these techniques use high-throughput DNA sequencing, coupled to the use of specific chemical reagents or specific antibodies to reveal modified RNA nucleotides.

  • An epitranscriptome analysis should be followed by thorough validation of obtained candidate sites by complementary orthogonal approaches.

  • Experimental and bioinformatic approaches and challenges for global analysis of RNA modification are discussed.

Abstract

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 as meaningful ways for validation.

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Figure 1: Reverse transcription-based techniques for detection of modified nucleotides.
Figure 2: Enrichment strategies in analysis of RNA modifications.
Figure 3: Library preparation issues.
Figure 4: Mass spectrometry approaches for validation of RNA modification.
Figure 5: Methods for validation of modified RNA nucleotides.

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Acknowledgements

This work was supported by joint Agence Nationale de la Recherche and Deutsche Forschungsgemeinschaft (ANR-DFG) grants for High-throughput technology for detection of RNA modifications (HTRNAMod) (ANR-13-ISV8-0001/HE 3397/8-1) and by the DFG priority programme SPP1784.

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Authors and Affiliations

  1. Institute of Pharmacy and Biochemistry, Johannes Gutenberg University Mainz, Staudingerweg 5, Mainz, 55128, Germany

    Mark Helm

  2. IMoPA UMR7365 CNRS-UL, BioPole Lorraine University, 9 avenue de la Foret de Haye, 54505, Vandoeuvre-les-Nancy, France

    Yuri Motorin

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  1. Mark Helm
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Correspondence to Mark Helm or Yuri Motorin.

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Glossary

RNA modifications

Chemically altered (modified) nucleotides in mature RNA chain.

Nucleotides

The smallest structural units of a nucleic acid, composed of a nucleobase connected to a ribose (or deoxyribose) via a glycosidic bond, and a phosphate esterified to a hydroxyl group of the sugar moiety. The combination of only nucleobase and sugar is referred to as a nucleoside.

Reverse transcription

The synthesis of single-stranded DNA (known as complementary DNA (cDNA)) by reverse transcriptase (an RNA-dependent DNA polymerase) using single-stranded RNA as a template.

RT signature

A specific pattern composed of misincorporation in cDNA and reverse transcription arrest of primer extension, induced by modified residues in an RNA chain.

Haptens

Small, otherwise immunosilent molecules that elicit an immune response only when exposed to the immune system in the form of a covalent conjugate to a large carrier such as a protein.

Pulldown

A technique for selective isolation of RNA (or protein) species from a complex biological sample. It generally relies on affinity capture with antibodies or another high-affinity interaction.

Click chemistry

A class of biocompatible (bio-orthogonal) reactions, generally involving azide and alkyne moieties.

Barcodes

Short specific sequences (often six to eight nucleotides) that are incorporated into sequencing adaptors and ligated to samples. When barcode sequences are shared within a sample but differ between samples, they allow unambiguous retrospective assignment of samples run in the same sequencing lane. When barcodes are degenerate and highly variable within a sample, they are known as unique molecular identifiers (UMIs) and are used to identify redundant, PCR-amplified reads that originate from the same reverse transcription event. Adaptors may combinatorially contain both sample-specific barcodes and UMIs.

CircLigation

Circularization of single-stranded cDNA templates having a 5′ phosphate and a 3′ hydroxyl group with CircLigase.

Receiver operating characteristics curve

(ROC curve). A graphical plot illustrating the performance of a binary classifier system as a function of varied thresholds for true positives and false positives. Plotting the true-positive rate (TPR) against the false-positive rate (FPR) at various threshold settings leads to the ROC curve, in which the area under the curve (AUC) is a measure of the performance. The true-positive rate is also known as sensitivity, the FPR can be calculated as 1 – specificity.

Lipophilicity

'Fat-loving' character of a compound, reflected in its ability to dissolve in nonpolar solvents.

DNAzymes

Artificially generated DNA sequences that contain catalytic activity, for example, RNase activity.

Thin-layer chromatography

(TLC). A technique for separation carried out in a thin layer of adsorbent material, also known as a stationary phase. TLC can be one-dimensional (1D-TLC) if it involves one type of separation or two-dimensional (2D-TLC) if it involves separation in one dimension using one solvent, followed by separation in a second dimension using a second solvent that separates on the basis of distinct physicochemical properties.

Splinted ligation

A technique to biochemically join nucleic acid fragments by enzymatic ligation. Both fragments are brought in spatial proximity by hybridization to a so-called splint DNA, which is an oligodeoxynucleotide that is essentially a cDNA of the target RNA molecule.

Nanopore technology

Technology that funnels molecules, including nucleic acids, through nanosized pores (typically protein-based pores), thereby opening the possibility of determining their sequence (and potentially also a subset of their covalent modifications).

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Helm, M., Motorin, Y. Detecting RNA modifications in the epitranscriptome: predict and validate. Nat Rev Genet 18, 275–291 (2017). https://doi.org/10.1038/nrg.2016.169

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