Investigating the molecular mechanisms of RNA silencing

RNA interference, commonly referred to as RNAi, is a new biological mechanism that allows scientists to artificially switch off genes. There is hope that this mechanism might be exploited in medicine, to switch off genes involved in disease. The major advantage of RNAi is that the main information required to design the drug, called a small interfering RNA, or siRNA, is the sequence of the gene to be silenced. This, in principle, would allow the treatment of many diseases which are currently untreatable. For example, siRNAs could be used to treat viral infections, such as influenza, SARS, or Herpes, or lethal neurodegenerative disorders, such as Huntingdon‘s disease. The research described here - termed structural biology - is aimed at producing molecular images of this biological mechanism. Knowledge of these molecular images, or molecular structures, would assist in the design of more effective siRNA drugs. This in turn would lead to better disease treatment. In addition, the research would throw light on the biology of stem cells, a type of basic cell that may in future be used for personalised tissue regeneration. This research is being carried out by Dr James Parker in the Laboratory of Molecular Biophysics at Oxford University.

RNA silencing refers to a group of recently-discovered gene silencing mechanisms mediated by short RNA molecules. The short RNA molecules, referred to as guide RNAs, act as the targeting components of the silencing machinery. Two forms of RNA silencing, RNA interference (RNAi) mediated by small interfering RNAs (siRNAs) and gene silencing mediated by genome-encoded microRNAs (miRNAs), operate by degrading or sequestering target mRNAs. The third main form of RNA silencing functions through the assembly of heterochromatin, thereby rendering genes inaccessible.

RNAi holds promise as a significant new form of therapy in the treatment of disease. The strategy is to use siRNAs to switch-off genes required for the progression of a disease. The prospect of workable RNAi therapeutic methods is tantalising. siRNAs of any sequence could be used, targeting any viral or human gene, thereby facilitating the treatment of infections or diseases which are at the moment untreatable. Significant progress has already been made using siRNAs to treat mouse models of respiratory disease (influenza virus, respiratory syncytial virus, parainfluenza virus, SARS), sexually-transmitted disease (Herpes simplex virus 2) and neurodegenerative disease (amyotrophic lateral sclerosis, spinocerebellar ataxia type 1 and Huntingdon‘s disease). Furthermore, RNA silencing pathways are required for the proliferation and differentiation of stem cells. MicroRNAs play key roles in the determination of tissue identity.

We propose to analyse the molecular mechanisms behind RNA silencing via structural biology. We would use X-ray crystallography techniques to determine the structures of key components and functional complexes within RNA silencing pathways. Specific objectives include 1) The determination of the structure of a guide RNA strand bound to Argonaute, the central component of the RNA silencing machinery, and a target messenger RNA, 2) The first structure of eukaryotic Argonaute, 3) The determation of two complexes involved in guide RNA processing: the helicase domain of Dicer, and a complex of TRBP with siRNA, and 4) The determination of the structure of the miRNA transporter, Exportin-5, in the presence and absence of miRNA. The structure of the guide-Argonaute-target co-complex would provide direct molecular information about the function of siRNAs in RNAi and therefore assist in the design of artificial siRNAs.

In summary, the projects offer the opportunity to gain insight into a significant new mode of gene regulation. The results would provide a foundation for more effective therapeutic RNAi and contribute to our molecular understanding of stem cell biology and the basis for tissue identity.