HTRNAMod

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.