Genome dynamics

MicroRNAs, a class of small non-coding RNAs known to act as post-transcriptional regulators, have recently been implied to play an important role in host:pathogen interactions, such as viral infections. While DNA viruses seem to express their own miRNAs in the host cell, only recently Hepatitis C Virus (HCV), a single stranded positive strand (+ss)RNA virus of the family Flaviviridae, was reported to necessarily depend on host cell miRNA-122 [1]. This is remarkable in that, while usual host cell recognition factors make sure the virus is in the right tissue, dependence on a cell state specific miRNA constricts virulence to a certain developmental or differentiational state of the cell. Prediction of RNA secondary structures in general and viral structures in particular has a long tradition at the TBI. We subjected the data reported by Jopling et al. [1] to our algorithms and observed (i) a slightly deferent binding site and (ii) quite dramatic changes in previously proposed structures within the HCV genome [2]. These preliminary data suggest that structural rearrangements propagate throughout the whole genome. Assuming that this mechanism might be conserved among other members of the family Flaviviridae, we applied our algorithms to tick-borne encephalitis virus (TBEV). A preliminary screen using all known human miRNAs (miRBase7.1) resulted in a few miRNA candidates, inducing genome dynamics similar to those observed in HCV. Thus, the following general questions arise: What happens to the structure of an RNA when some other RNA binds to it? Given structural changes are induced, how do the resulting RNA dynamics influence the function of the RNA? And finally, are such regulatory systems evolutionary conserved and thus a general principle or unique innovations of individual species? Previous work focused on detection, description and evolutionary conservation of RNA secondary structures in viral RNA genomes. Therefore we want to proceed to the next steps outlined as follows: 1 screening for miRNAs interacting with genomes of RNA viruses, e.g. Flaviviridae 2 development of algorithms for genome wide kinetic folding simulation 3 experimental verification of position and function of predicted miRNA:viralRNA interactions 4 elucidation of the role of miRNA:viralRNA interaction with regard to translation, replication, and packaging of the viral genome References [1] CL Jopling, M Yi, AM Lancaster, SM Lemon, and P Sarnow. Modulation of hepatitis C virus RNA abundance by a liver-specific microRNA. Science, 309(5740):1577-1581, Sep 2005. [2] C Thurner, C Witwer, IL Hofacker, and PF Stadler. Conserved RNA secondary structures in Flaviviridae genomes. J Gen Virol, 85(Pt 5):1113-1124, May 2004.

A relatively new concept, on how cells manage to coordinate a large amount of different chemical reactions as well as physiological and regulatory events within the close spacial proximity of a cell compartment, was the finding of micro RNAs (miRNAs). These are very small RNA molecules of 19 to 22 nucleotides length, which are able to silence mRNA expression very efficiently within very short periods of time. The process of cutting out and processing them is initiated by an enzyme called Drosha, which resides in the nucleus of the cell. It is also common knowledge, that also viruses, which are generally considered as free genomic elements, explore this convenient way of interacting with their host cells as well. A subgroup of viruses, however, which stalls its genome not in the form of DNA, but as RNA, and which has no DNA stage in its life cycle, was so far believed to be excluded of this system. One central goal of this project was to show, that members of the viral family flaviviridae indeed could interact by miRNAs with their hosts. We could introduce an artificial miRNA into the genome of the virus and detect functional miRNAs in the host cell. This has significant impact in both fields, in the further research on the miRNA processing pathway and/or RNA viral life cycle, as well as in the development of new vaccines and vectors. We characterized a special region within the viral genome, which is highly tolerant to alterations, and which can be used to introduce genetic information into host cells upon viral infection. An other aim of this project was, to study the structural behavior of large RNA molecules. Single stranded RNA molecules have the capacity, to refold onto themselves and by doing so build short double helical structures, so called stem-loops, which may be recognized of enzymes and thus exert important functions. Yet one single RNA molecule may form a large variety of structures, which could not be found by simple minimum free energy calculations. We managed to implement an algorithm which allowed us to calculate folding pathways of RNA molecules of up to 1500 nucleotides length. This comprises almost all structurally relevant RNA molecules in the cell and can help to understand their structural dynamics. We now can find structural traps, which retard or even prevent molecules to reach their thermodynamically favorable structure. We now have a tool at hand, which helps us observe, in which state during the folding process certain regions of the molecule are accessible to other molecules, and when they are hidden in a secondary structure and thus not recognizable to other enzymes in the cell.