[
International Worm Meeting,
2021]
We are interested in understanding the protein and RNA components of the spliced leader trans-splicing machinery. Previous work has implicated a set of novel non-coding RNA components, the SmY RNAs, in spliced leader trans-splicing. These RNAs are encoded by 12 distinct genes and we are in the process of systematically deleting them to understand the precise mechanistic roles of their products. To facilitate this approach we have developed a novel hph::gfp reporter transgene that allows tagging and knockout of any gene of interest in a single injection via CRISPR/Cas9 induced homology directed repair (HDR). This method generates a loss of function allele and a fluorescent protein fusion reporter, while the hph hygromycin resistance gene facilitates the selection process. The fusion protein is functional, successfully generating both broad cytoplasmic fluorescence and hygromycin resistance in the assayed worms. This hph::gfp repair template provides a simple and flexible approach - it can be modified by oligo cloning to be flanked by homology arms corresponding to the region upstream and downstream of any gene of interest. This approach significantly reduces the time and labour required to achieve each knockout, facilitating our goal of complete smy gene complement knockout. Our results to-date show that loss of ten of the twelve smy genes impair spliced leader trans-splicing, confirming the role of this enigmatic set of RNAs in this process.
Connolly, Bernadette, Fasimoye, Rotimi, Wenzel, Marius, Eiljers, Peter, Soto-Martin, Eva, Pettitt, Jonathan, Muller, Berndt, Elmassoudi, Haitem, Spencer, Rosie
[
International Worm Meeting,
2021]
We are investigating spliced leader (SL) trans-splicing and its key RNA and protein components as potential anthelmintic targets, using Caenorhabditis elegans as a model system. SL trans-splicing is an essential process in nematode gene expression that facilitates translation by replacement of the 5' untranslated region of most mRNAs with the spliced leader 1 (SL1). The splicing reaction involves an interaction between the SL1 snRNP, the nascent pre-mRNA and the spliceosome. Although SL trans-splicing was discovered more than 30 years ago, we know little about the molecular mechanism(s) by which this is achieved. To address this, we have carried out a comprehensive molecular characterisation of the SL1 snRNP. This work expands and refines our understanding of the proteins involved in SL1 trans-splicing: we have analysed factors co-immunoprecipitating with the SL1-specific protein SNA-1, giving us insight into the interaction of the SL1 snRNP with the spliceosome. Proteins critical for SL1 trans-splicing were identified using established RNAi-based qPCR and gfp-reporter gene assays (https://doi.org/10.1093/nar/gkx500). This led to the identification of a novel, essential trans-splicing factor termed SNA-3. SNA-3 is a highly conserved, nematode specific protein containing NADAR domains, which have been linked to NAD/ADP-ribose metabolism and may have N-glycosidase activity. SNA-3 interacts with several highly-conserved proteins associated with RNA processing including the CBC-ARS2 complex components NCBP-1 and SRRT/ARS2 involved in co-transcriptional determination of transcript fate. Together, these observations implicate SNA-3 in key steps linking SL1 trans-splicing to the transcriptional control of gene expression. The identification of another essential, nematode-specific protein involved in SL1trans-splicing strengthens the hypothesis that the acquisition of SL trans-splicing requires the evolution of novel machinery required to modify the activity of the spliceosome. The novelty of these proteins makes them ideal targets for the development of new anthelmintics.