An effort to elucidate mechanisms of the RNA interference pathway in Trichomonas vaginalis

Abstract

One of the most prevalent sexually transmitted infections in the world, trichomoniasis, is caused by the microaerophilic protozoan Trichomonas vaginalis, which is found in the human urogenital tract. T. vaginalis has an unusually large genome (over 160Mb) for a unicellular eukaryote which was published in 2007. It can be potentially used as a model organism to study the early evolution of eukaryotes. However, despite its significance for both medicine and academia, T. vaginalis is not given enough attention around the scientific community. The T. vaginalis genome remains poorly annotated, and little is known about the regulatory mechanisms necessary for gene expression. RNA interference (RNAi) is a regulatory mechanisms which can significantly reduce the expression of a gene without removing it from the genome. RNAi is thought to have evolved in all eukaryotes and has emerged as a useful tool to study gene function. Several deep-branching protozoa have all been found to exhibit RNAi activity. In T. vaginalis, there have been sporadic reports of double stranded RNAs suppressing gene expression in T. vaginalis. It has been shown in the Barbosa group that short hairpin RNAs (shRNAs) are potent suppressors of gene expression. In this study, through the construction of 20 small RNA or RNAi-expressing plasmids and the use of digital droplet PCR, we were able to highlight the effectiveness of small RNAs for gene knockdown in T. vaginalis. We also showed that by altering the structure of the shRNA molecule, knockdown effectiveness can be increased significantly. Notably, a 34nt antisense RNA construct produced extraordinary effectiveness in knocking down gene expression of the highly expressed ferredoxin gene in T. vaginalis. Lastly, we demonstrated the possible nuclear cytolocalisation of the T. vaginalis Argonaute two protein through indirect-immunofluorescence. Collectively, this work provided new insights and tools for the study of gene expression and function in T. vaginalis. Most importantly, this work provided evidence for potential Dicer-independent RNAi activity in T. vaginalis and the possibility of dsRNAs not being required for the purposes of gene knockdown in this unicellular parasite.