High-throughput technology for detection of RNA modifications – HTRNAMod

In order to analyze numerous modified nucleotides presents in RNAs, we are using high-throughput DNA sequencing technology (so called Next-Generation DNA Sequencing – NGS). In odrer to detect RNA modifications, the RNAs are first treated with an appropriate chemical reagent, for selective derivatization of certain modified residues only, without affecting normal nucleotides or other modifications. These derivatized nucleotides will serve as road-blocks for primer extension by reverse transcriptase. The cDNA products are thus converted to double stranded DNA fragments and amplified using barcoding oligos for sequencing. The resulting amplicons are sequenced using fluorescent terminators using Illimina sequencing machines. The data obtained are used for alignment to the reference sequence followed by analysis of positions of read's extremities. By comparing treated/untreated profiles the presence of the modified residue can be detected.

At the beginning of the project, we established all the required techniques and procedures for sample preparation for sequencing, these methods allow precise detection of RT stops in RNA, as well as the misincorporation of nucleotides at a given position. This approach was first applied to detection and mapping of modified m1A residues in different tRNAs, and we even demonstrated the presence of these modified nucleotides in Trypanosome.
Now, to extend the repertoire of RNA modification detectable by high-throughput sequencing we established the experimental protocol for detection of 2'-O-methylations. This method is based on the resistance of the phosphodiester bond when the adjascent nucleotide is methylated at its 2'-OH. The developed approach is now used in the lab for profiling of 2'-O-Me variations in different cell lines and in pathologies.
Currently, other methods combining specific chemical treatment with high-throughput sequencing are under progress, notably for detection of m7G and m3C.

The development of approaches requiring the detection and localization of modified nucleotides in RNA is now attracting wide interest. For the moment, out of more than 150 known RNA modifications, only 5 or 6 are detectable at a large scale and globally in the transcriptome. These methods are still limited only to ubiquitous RNA modifications, present in many different organisms. On the other hand, development of such approaches is related to the availability of specific chemical reagents for specific treatment of modified nucleotides. As future developments, we can envisage the extension of these methods for more and more different RNA modification types. In addition, one can expect applications of these innovative technologies to study regulation and de-regulation of RNA modifications in human pathologies and metabolic dysfunctions.

Bourgeois, G., Ney, M., Gaspar, I., Aigueperse, C., Schaefer, M., Kellner, S., Helm, M., and Motorin, Y. (2015). Eukaryotic rRNA Modification by Yeast 5-Methylcytosine-Methyltransferases and Human Proliferation-Associated Antigen p120. PloS One 10, e0133321.
Hauenschild, R., Tserovski, L., Schmid, K., Thüring, K., Winz, M.-L., Sharma, S., Entian, K.-D., Wacheul, L., Lafontaine, D.L.J., Anderson, J., et al. (2015). The reverse transcription signature of N-1-methyladenosine in RNA-Seq is sequence dependent. Nucleic Acids Res. 43, 9950–9964.
Tserovski, L., Marchand, V., Hauenschild, R., Blanloeil-Oillo, F., Helm, M., and Motorin, Y. (2016). High-throughput sequencing for 1-methyladenosine (m1A) mapping in RNA. Methods (Elsevier) in press.

Mature RNAs contain numerous modified nucleotides which are all formed post-transcriptionally, by specific action of dedicated enzymes. While our knowledge is mostly limited to highly abundant and stable RNA species like tRNA, rRNA and snRNA, only fragmentary data is available on the presence and precise localization of RNA modifications in other cellular RNAs, like snoRNAs, small regulatory RNAs and mRNAs. Recent developments brought a new dimension in the understanding of RNA modification roles and functions in the cell, invoking an epigenetic character of RNA modifications. These question the established concept of a stable, durable, and “life-long” character of RNA modifications. Evidence supports the idea of RNA modification as a general regulatory and therefore transient phenomenon, potentially of an importance equal to alternative splicing or A-to-I editing. Recent top-ranking papers clearly identified regulated as well as regulatory RNA modifications. In addition to mRNA, this also applies to the supposedly concrete-cast tRNA modifications, and an extension to regulatory RNA is easily anticipated. Despite the key importance of these modifications in regulation of the cellular metabolism, little is known on their presence in different cellular RNAs and their exact localization. The recent breakthroughs are limited to certain modifications and have been enabled by the use of either RNA Seq or mass-spectrometry approaches. There is consensus among specialists, that the current bottlenecks in this field are twofold, namely: (i) the detection of the chemical structure of modifications in a given RNA and (ii) the mapping of their localizations. Currently, the analysis of individual RNA molecules is a difficult and laborious task, which is limited by the requirement for high amounts of starting material. Hence, this proposal aims at the development of new technologies allowing high-throughput analysis of RNA modifications. As a central strategy, we will combine two current principles that are available for high- throughput detection of modifications, namely selective chemical transformation and reverse transcription (RT) arrest. RNA with known modifications will be treated with various agents known or suspected to chemically transform the modifications such as to alter their behaviour during RT. Such altered behaviour will be used to define an RT-signature from RNA Seq data of known modifications, which will then identify candidate sites in transcriptome-wide RNA-seq data. For validation, RNA species hosting candidate sites will be isolated by robot-assisted technology in quantities sufficient for LC-MS/MS analysis. The latter will be applied to confirm the existence of predicted modification at candidate sites. The goal of the project is to provide tools for whole-transcriptome analysis of modifications in RNAs, with the further extension of such analysis to global changes of RNA modification pattern in normal development and pathologies.