This project is an interdisciplinary approach to basic and applied research on RNAs with tRNA properties. It was divided into four inter-related parts. The first dealt with recognition of RNA domains by tRNA-recognising proteins, the second concerned RNA and tRNA-like domains from viral RNA genomes, the third comprised selection and structure analysis of RNA domains mimicking tRNAs, and the fourth dealt with biotechnological aspects of RNA chemical synthesis.
The feasibility of the project relied on the complementarities between the participating teams in terms of expertise in methodologies (RNA engineering, combinatorial methods, X-ray crystallography, NMR, RNA chemistry, and bio-computing) and availability of a variety of proteins from translation and replication systems.
The RNAs that were investigated derived from canonical tRNA (taken as a model) or viral tRNA-like structures. They were obtained either by rational structure-based design or by in vitro selection. A major aim was to understand detailed molecular aspects of the translation machinery, but transcription / replication processes were also considered since tRNA or tRNA-like domains participate in such processes. The final aim was to find small RNAs that specifically block tRNA-target proteins (tRNA maturation endonucleases, aminoacyl-tRNA synthetases, elongation factors, viral replicates), with the hope that our project should lead in the future to the design of new classes of RNA antibiotics and to the proposal of novel anti-viral strategies.
A variety of steps in translation (some in replication) were therefore studied and experiments were carried out on RNAs of various biological origins [viruses, prokaryotes (mesophiles or thermophiles), eukaryotes, archaea]. Species specificities between these RNAs, in particular for aminoacylation and elongation, were investigated. The problem of evolution was approached by theoretical methods for the search of RNAs with tRNA characteristics and by a variety of experimental methods including particular in vitro selection procedures. In this way we expected to find RNA structures similar to those retained by natural evolution, but our hope was also to find alternate solutions providing aminoacylation identities or recognition potential by translational factors.
An extremely positive aspect of the project after two years of activity was the establishment of many links between partner Laboratories. Some already existed but have been rejuvenated [e.g. between Team 1 (Strasbourg) and Teams 2 (Illkirch), 3 (Leiden) and 5 (Bayreuth) or between subcontractor of Team 1 and Team 7 (Göttingen)] and more important, many new ones were created. Their birth and the start of actual collaborations were greatly facilitated by the joint and bilateral Meetings and Workshops that were organised in the frame of this contract. Collaboration between Team 3 (Leiden) and Team 4 (Vienna) initiated work for RNA structure prediction and especially for that of metastable structures. Other cooperatives were between the Vienna Team and Team 1 (Strasbourg), or Team 5 (Bayreuth) and Team 6 (Rome). For that, a number of RNA structures were provided to the Vienna Team, either wild-type or mutant sequences, for prediction of their most likely folding. Other links, related to common interest in RNA-aptamer research, connected Team 5 (Bayreuth), Team 6 (Rome), and Team 1 (Strasbourg). Cooperations involving the industrial partner (Team 7, Göttingen) were particularly fruitful: they concerned chemical synthesis of modified RNA blocks and tRNA fragments with modified bases (with Team 1, Strasbourg), synthesis of fluorescent nucleotide or dinucleotide analogues (with subcontractor of Team 1, Strasbourg and Team 5, Bayreuth), and synthesis of RNA fragments for studying pseudoknot structures (with Team 3, Leiden)