Analysis of spliced-leader trans-splicing in C. elegans and the identification of novel targets for the development of new anthelmintics

Abstract

The spliced-leader (SL) trans-splicing is essential for gene expression and the viability of nematodes. The 5’ end of the pre-mRNA in the SL trans-splicing, called the outron, is replaced with a short RNA called the SL in a reaction that is mechanically similar to intron removal by cis-splicing. The molecular machinery executing this process is poorly understood. To resolve this, we have developed a green fluorescent protein (GFP)-based assay which identifies the spliced leader trans-splicing inhibition in a viable Caenorhabditis elegans (C. elegans). The assay is used in an RNAi screen to identify new genes involved in the SL transsplicing. I checked 2446 individual genes located on chromosome I and found 3 genes that potentially encode novel components of the trans-splicing machinery. RNAi treatments targeting genes ncbp-1, ncbp-2 and teg-4 all activate the reporter gene, suggesting a role in spliced leader trans-splicing. I confirmed the role of the three genes in this process by a reverse transcription quantitative polymerase chain reaction (RT-qPCR) to have an impact on SL1 trans-splicing as the knockdown of the genes increased outron retentions for both gfp and rps-3 transcripts. As a future work, I decided to look at identifying novel components of the pathway that might could be suitable targets for drugs development. I want to further confirm the impact of the genes, targeted in our research, on SL trans-splicing using immunoprecipitations and proteomic approaches