Understanding the mechanism of a nematode molecular Achilles' heel.

Parasitic nematodes are major challenges to human health and global food security. The impact of these animals is exacerbated by drug resistance, withdrawal of existing treatments due to environmental concerns, and expansion of parasites into new environments through climate change and human migration. There is thus an urgent need to develop new control methods. Ideal targets for the development of new therapeutics would be molecules and processes that are found only in nematodes and are absent from the animals and plants that they infect. Ideally these targets would be found in all nematodes, enabling the development of a drug active against a broad range of nematode infections. One such process is spliced leader trans-splicing, which is an essential step by which many nematode genes become active.

We have shown that two 'molecular machines' are required for spliced leader trans-splicing. Nematodes in which these machines are non-functional stop developing and are infertile. However, we don't have the detailed knowledge of these machines needed to understand how they function, a necessary precondition to properly develop drugs that inhibit them. In particular, we don't know all the components involved, and we don't understand those that we do know about. To address these challenges, we are systematically studying the components of these machines in the experimental nematode Caenorhabditis elegans. Our work so far has led to the identification of a new component which has properties that make it an ideal potential drug target.

The proposed research will extend our initial studies, to give a comprehensive picture of the molecular machinery that underpins this essential step in nematode gene expression. The process of spliced leader trans-splicing has evolved multiple times in different groups of organisms, many of which are also important parasites. Thus, as well as allowing the development of potential new drugs to treat nematode infections, our findings will serve as a starting point to investigate the similarities and differences between other 'versions' of trans-splicing.

Spliced leader trans-splicing is an essential gene expression step in many eukaryotes. It occurs by modification of the spliceosome to facilitate the intermolecular reaction between short, non-coding 'spliced leader' RNAs and their target pre-mRNAs. This results in addition of the spliced leader onto the 5' end of the mature mRNA. In nematodes, the majority of trans-splicing events depend on the SL1 and SmY ribonucleoproteins. However, we do not know how these complexes work, nor do we have complete knowledge of their compositions. To address these knowledge gaps, the proposed research will:

1. Determine the molecular composition of the two ribonucleoproteins critical for nematode SL1-type trans-splicing.

2. Define the activity, molecular determinants and structure of SNA-2, the essential protein component of the two ribonucleoproteins.

3. Understand the function of SNA-3, a newly identified essential component of the trans-splicing machinery.

Our research programme combines advantages of the model nematode C. elegans with advanced analytical techniques, including protein identification by mass spectrometry, next generation sequencing and structure analysis by X-ray crystallography and NMR spectroscopy. Validating a key approach - identification of ribonucleoprotein components by immunoprecipitation followed by mass spectrometry - we have identified SNA-3, a new protein involved in trans-splicing. We are in an excellent position for the successful completion of this programme that will provide detailed information about the molecular composition of ribonucleoproteins involved in SL1 trans-splicing, and the molecular function of the key components.

Based on our recent work, which showed trans-splicing is conserved throughout the nematode phylum, this research will have broad impacts on our understanding of nematode biology and mRNA splicing, and inform our understanding of gene expression in other organisms that employ this process.

Findings from this work will impact on the work of researchers that study gene expression in nematodes, and of researchers studying gene expression in a wide range of other eukaryotic organisms. It will inform the work of researchers studying mRNA splicing in other organisms and may impact on researchers working in the areas of synthetic biology and gene therapy.

Our work will impact upon the UK science skills base, and its concomitant effect on the country's economic competitiveness. The project helps address skill deficits in RNA splicing and fundamental biochemistry identified in the "BBSRC and MRC review of vulnerable skills and capabilities". The research workers employed on the project will benefit from training in the research specific skills involved. These skills will be further disseminated throughout the researchers' subsequent careers, ultimately benefitting the wider research base.

Since nematodes are major parasites of humans, livestock and economically important plants, some of the main beneficiaries will be commercial and non-commercial enterprises with an interest in the development of drugs to treat parasitic infections (anthelmintics). Our research is focussed on understanding an essential, conserved nematode gene expression process. Understanding this process will provide novel, nematode-specific targets for the development of new anthelmintics. The need for these drugs in the treatment of livestock infections is acute due to the global incidence of anthelmintic resistance, even to the most recently developed drug, monopantel. However, plant parasitic nematodes represent some of the greatest challenges to global crop production: almost every cultivated crop, for instance, is prone to infection by root knot nematodes. Previous treatments to control plant parasitic nematodes have been withdrawn due to their environmental impacts, so there is increased need to identify new compounds to treat these nematodes.

Impact will be achieved through publication of research findings, deposition of data in relevant archives, and plasmids and C. elegans strains that will be made through appropriate resource centres.

Finally, we plan to increase the societal impact of our research through a series of public engagement activities. Dr Pettitt has extensive, prize-winning expertise that covers a broad range of engagement events. We will exploit the skills and connections that he has previously established to raise awareness of the ecological importance of nematodes, and of their importance for animal welfare and food security.