NMR view of tRNA maturation – NMR-VitAmin

In-cell NMR represents a novel biophysical tool to investigate chemical and structural modifications of biomolecules at the atomic level inside living cells. Indeed, in vivo and/or ex vivo NMR has recently achieved great methodological advance. For instance, remarkable results have been obtained by monitoring the incorporation of post-translational modifications in proteins within a cellular context or even in living cells using the microinjection of the protein of interest into Xenopus oocytes. These studies have demonstrated that in-extract and in-cell NMR are particularly well suited to monitor the maturation process of biological macromolecules, like proteins. The development of in-cell NMR with nucleic acids is equally challenging. Exploring further along the same lines, our project aims at deciphering tRNA maturation events with the same powerful in-cell NMR technology.

We have already obtained a number of results, which demonstrate the technical feasibility and the pertinence of the study of tRNA maturation by in vivo NMR. Indeed, these experiments show that:
(i) tRNA can be produced by in vitro transcription in mg quantities and easily purified by chromatography;
(ii) modern NMR spectrometers with cryogenic probes allow measurement of tRNA spectra of high quality in cell-extract, even at low concentrations (50-100 µM). They also allow measurement of tRNA spectra in living cells like Xenopus oocytes;
(iii) tRNAs are stable RNA that are not rapidly degraded in cell-extracts or in-cells;
(iv) the kinetics of incorporation of post-transcriptional modifications are slow enough to be observable by a series of 2D NMR spectra.
Judicious isotopic labelling strategy of tRNA and co-factors together with the implementation of new NMR experiments allowed us to monitor the maturation of tRNAs in cellular extracts by NMR (results to be published).

This challenging project is very innovative both for the NMR field of RNA and for the (t)RNA communities. tRNAs are important functional molecules in all cells. Overall, tRNA modifications have been for a long time believed to be implicated almost exclusively in the decoding step of translation and in the stabilization of the three-dimensional architecture of tRNAs. However, the idea that tRNA modifications and their respective pathways can be connected with regulatory functions has recently emerged. Indeed, new roles of tRNA modifications as connectors between translation, metabolism and stress response have been recently uncovered. It became clear that tRNA modifications are central to a global quality control of the cell integrity and that tRNA maturation pathways might be placed at the cross-road of various regulation pathways such as the response to oxidative stress or metabolite starvation. The methodology described in this project could pave the way to the molecular study of such pathways.

1. Poster at SifrARN meeting (8-10 mars 2016) «The maturation of tRNAs investigated by in cell NMR spectroscopy.»

In-cell Nuclear Magnetic Resonance spectroscopy (in-cell NMR) represents a novel biophysical tool to investigate structural transformations of biomolecules at the atomic level inside living cells. Indeed, NMR in vivo and/or ex vivo has recently undergone great advances, and remarkable results have been obtained by monitoring incorporation of post-translational modifications in proteins within a cellular context. These studies demonstrated that in-extract and in-cell NMR are particularly well suited to monitor the maturation process of biological macromolecules, such as proteins. Along the same lines, our project aims at deciphering RNA, mostly transfer RNA (tRNA) maturation events with in-cell NMR. The genomic and the improvements of analytical tools have led to a renaissance of the tRNA modification field. Some human diseases have now been clearly linked to the absence of tRNA modifications. The role of tRNA modifications as regulatory mechanisms needs to be studied further. In addition, many of the in vivo roles of tRNA modifications remain undisclosed. This proposal aims at deciphering the maturation process of tRNA at an atomic level, including information on the chronology of the events leading to the biosynthesis of a fully mature and functional tRNA. The originality and the challenge of this proposal entitled “NMR View of tRNA maturation” (NMR-VitAmin) resides in the use of in-extracts and/or in-cell NMR to elucidate some critical aspects of tRNA biosynthesis and maturation under the conditions of a living cell. NMR will be also used as a screening tool in-extracts to evaluate the action of an RNA-binding chemical library on the tRNA maturation process. Actually, we also aim at interfering with this highly regulated process, in which a misbalance in the level of tRNA modifications is often associated with pathologies.