Les nouvelles fonctions catalytiques et régulatrices de l’ARN chez les eucaryotes / The new catalytic and regulatory functions of RNA in eukaryotes

Séminaire organisé par Eric Westhof du 10 au 15 Mars 2008

Participants

Jérôme Cavaillé, Bernard Dujon, Witold Filipowicz, Daniel Gautheret, Christian Hammann, Tan Inoue, John Mattick, Heinrik Nielsen, Sébastien Pfeffer, Mikez Pheasant, Pascale Romby, Renée Schroeder, Olivier Voinnet, Gerhardt Wagner, Eric Westhof (organisateur), Sarah Woodson, Mihaela Zavolan

Résumé en français

Des travaux récents sur des plantes, des champignons et des animaux ont révélé de nouveaux mécanismes de contrôle génétique conservés au cours de l’évolution.  Les régulateurs sont de petits ARN longs de 21 à 25 nucléotides obtenus après  maturation soit d’ARN double brin exogène soit d’ARN endogène transcrit à partir de régions intergéniques ou répétitives. Les premiers sont appelés ARN interférents et les seconds microARN. L’interférence de l’ARN constitue un formidable outil d’analyse fonctionnelle génomique. Au niveau fondamental, les rôles régulateurs des microARN interviennent dans de nombreux aspects de la vie cellulaire, de la virulence des pathogènes aux mécanismes épigénétiques des eucaryotes.  Les évidences s’accumulent pour montrer que les génomes sont entièrement transcrits. Par contre, moins de 2 % du génome humain code pour des protéines. Où et comment interviennent les nombreux transcrits produits ? Comment repérer et identifier les transcrits les plus importants dans un génome ? Voici quelques-unes des questions que nous avons discutées. L’extrême diversité des fonctions régulatrices de l’ARN, aussi bien chez les procaryotes que chez les eucaryotes, qui en se liant soit à des protéines, soit à d’autres ARN, soit à des métabolites secondaires, soit en ayant une action catalytique sur eux-mêmes ou sur d’autres ARN dévoile une complexité sous-jacente inattendue et promet de mener à des nouvelles stragtégies thérapeutiques dans le futur.

Compte rendu (en anglais)

The days are gone where RNA was solely the transcript of genetic information and as tRNA the carrier of amino acids to the ribosome, in itself a RNA-protein complex, for peptide bond formation. The discovery, in the early eighties, by Sidney Altman and Thomas Cech of catalytic RNAs changed entirely our views on RNA.

This breakthrough led to a fantastic upsurge in activity on RNA with the development of new techniques and an enormous increase in knowledge about RNA folding and activity. First, Darwinian Evolution in vitro, Systematic Evolution by Exponential Enrichment of ligands (SELEX) invented in the late eighties by Jack Szostak, Andy Ellington and Gerald Joyce lead to novel RNA molecules with chosen activities, the aptamers, and to precious views on the amazing power of selection with memory at the molecular level. Nowadays, tiny RNA molecules, microRNAs (miRNA) and small interfering RNAs (siRNA), are transforming our thinking on genetic regulatory processes. Most of the scientists involved in the deciphering of interfering RNAs started their careers, as chemist or molecular biologist, studying catalytic RNA (e.g. David Bartel or Thomas Tuschl). Witold Filipowicz spoke about the mechanisms and reversibility of the microRNA-mediated repression in mammalian cells. OlivierVoinnet described the post-transcriptional RNA silencing pathways in plants and their modulations by pathogens. Sébastien Pfeffer opened new mechanisms in the role of microRNAs in viral infections. Jérôme Cavaillé approached epigenetics and presented new data on imprinted small noncoding RNA genes. John Mattick revealed the hidden layer of regulatory RNA in the development of complex organisms and opened widely the central role and importance of RNA molecules in life development. However, as stressed by several speakers, the biochemical pathways explaining how the microRNAs regulate the translation of mRNAs is still poorly understood.

For a long time, x-ray crystallography of RNA was very difficult. The striking realization of the catalytic potential of RNA pushed chemists to develop new strategies for the synthesis of RNA molecules of definite composition and length. Similarly, crystallographers faced energetically the problems of RNA crystallization. This research front led, at the structural level, to the crystallography of the two subunits of the bacterial ribosomes at high resolution in the late nineties, a feat which has increased by orders of magnitude the data base of RNA structures and motifs. The pathway from a sequence to a functional structure is not yet fully appreciated and Sarah Woodson presented new views on the folding of RNA in the test tube and the cell.

At the same time, progress in sequencing full genomes has led to a vast amount of available sequence data. RNA  structural bioinformatics is based on comparative analysis of sequences. The challenge nowadays resides in the integration of the rich and complex 3D information gained by crystallography within the ever increasing databases of sequences. The implications are that systematic comparisons between crystal structures and aligned sequences should tease apart the key molecular connections maintaining a biologically functional RNA from the inescapable contingency inherent to any isolated sequence. Bernard Dujon introduced a few themes on eukaryotic genome evolution focussing on yeast genomes. Eric Westhof, exploiting sequence data and comparisons with crystal structures, stressed the observation and our present realization that neutral evolution is embedded within non coding RNAs. On the bioinformatic side, Mihaela Zavolan described new computational methods for miRNA gene and target discovery, while Mike Pheasant illustrated the difficulties of searching for non coding RNAs in mamalian genomes. In the procaryotic world, Daniel Gautheret proposed a functional classification of microbial non-coding sequences using phylogenetic profiling.

Several discoveries have now demonstrated that RNA is a multifunctional molecule performing different tasks in nature as ribozymes, as riboswitches or in RNA interference, or by selection as aptamers or artificial ribozymes. Renée Schroeder linked both genomics and selection for the experimental search  of new RNAs by developping genomic SELEX for the identification of RNAs that regulate transcription

The realization of the catalytic potential of RNA strengthened the hypothesis about the role of RNA at the origin of life four billion years ago. The new results lead us to suspect not only that DNA is a modified RNA but also that its functions are under control of tiny RNA molecules. As cellular complexity rises, the roles of noncoding RNAs increase and diversify. For example, recently, a new an unsuspected functional role for a catalytic ribozyme was discovered in which the formation of a microlariat mimicks the cap structure of eucaryotic messenger RNAs. Heinrik Nielsen described the ribozyme components of such twin-ribozyme introns, while Christian Hammann revisited elegantly the small endonucleolytic ribozymes, often hidden in unsuspected cell lines with unknown functions still to be discovered.

In conclusion, RNA molecules exert their regulatory power on gene expression in many ways. Some mRNAs develop regulatory elements with catalytic activity under allosteric control by small molecules. Ribozymes with well-characterized catalytic mechansims acquire alternative catalytic possibilities which are used in the stability of mRNAs. Thus, globally, regulation does not occur solely by cis or trans complementary Watson-Crick base pairing between RNAs. The catalytic power of RNA is also exploited in regulation processes.  Not only is every single step in the maturation process of a mRNA a site of potential regulation  (e.g. alternative splicing, 3’ end maturation, …) but new pathways are constantly discovered. Detailed phenomena are being deciphered in bacteria. Thus, Gerhardt Wagner explained the regulatory RNAs in bacteria and their biological roles and mechanisms, while Pascale Romby focused on the Staphylococcus aureus regulatory RNAs and how they contribute to virulence and adaptation in diverse environmental conditions. Finally, Tan Inoue introduced synthetic biology and the potential of its applications in the RNA field.