Efforts to elucidate functional and regulatory aspects of gene expression in Trichomonas vaginalis

Abstract

Trichomonas vaginalis is a microaerophilic protozoan that resides in the human urogenital tract causing trichomoniasis, a sexually transmitted infection worldwide. It is also an organism of interest for studying the early evolution of eukaryotes. Despite its medical and academic importance, the T. vaginalis genome, published over a decade ago, remains poorly annotated and little is known about the regulatory mechanisms required for gene expression. This study aimed to develop tools and methods that could facilitate functional genetic studies of T. vaginalis and help elucidate the processes of translation and capping in this parasite. Genome-wide expansions of gene families and the intrinsic oxygen sensitivity of T. vaginalis impede the application of many genetic tools to its biological research. To address this issue, in the first part of my study, I explored suitable molecular tools for this protist. First, I showed that iLOV, an oxygen-independent fluorescent protein from Arabidopsis thaliana, outperforms the conventional enhanced green fluorescent protein. With a faster maturation rate and stronger fluorescence intensity, iLOV can better serve as a reporter to investigate ongoing cellular activities and quantify gene expression level in T. vaginalis. Next, I demonstrated that the strong RNA polymerase (Pol) III promoter of the U6 snRNA gene in T. vaginalis exhibits a similar structural organisation to the orthologues in metazoans and can be used to produce sequence-customisable non-coding RNAs in this protist. This finding contributed to the successful deployment of the gene editing technology CRISPR/Cas9 in T. vaginalis for the first time. Finally, having this promoter and the iLOV reporter, I was able to develop a stable expression system for short hairpin RNAs, capable of downregulating gene expression in a sequence-specific manner. This system is not only superior to earlier gene silencing strategies for T. vaginalis but it also brings insights into the existence of an RNA interference pathway in this evolutionarily unique eukaryotic organism. A majority of protein-coding genes in T. vaginalis are controlled by two core promoter elements, which generate mRNAs with distinctively short 5′ UTRs. The specific mechanisms adopted by T. vaginalis to fine-tune the translation efficiency (TE) of these unique transcripts remain largely unknown. Using the available quantitative omics data of this organism, I examined the roles of codon usage bias and RNA secondary structures in TE regulation of T. vaginalis mRNAs. I then designed a synthetic library of synonymous iLOV genes and expressed them in T. vaginalis. The fluorescence intensities produced by the in vivo expression of these genes validated my computational predictions. T. vaginalis U1, U2, U4 and U5 spliceosomal snRNAs remain as the only cases of eukaryotic Pol II transcripts that do not receive a canonical guanosine cap co-transcriptionally. This fact makes RNA capping in T. vaginalis an interesting research topic, which was investigated in the last part of this study. I initially observed that the four Pol II snRNAs generally have high structural complexities at their 5′ ends, different from the folding patterns of the capped mRNAs and snoRNAs in T. vaginalis. Hence, using U2 snRNA as a model, I investigated the possible role of the stem-loop structure formed during its early transcription in the regulation of capping. I hypothesised that disruption of this structural motif may restore the guanosine cap, because similar phenomena have been observed for human and yeast U6 snRNAs. Two mutagenesis schemes were designed to disrupt the intramolecular base-pairing within the nascent U2 transcript and the structural variants were expressed in T. vaginalis. An examination on their 5′ ends indicated that the U2 transcripts remained uncapped, which did not support our hypothesis. Therefore, factors that preclude spliceosomal snRNAs in T. vaginalis from receiving a guanosine cap may come from the transcription machinery, instead of the RNA sequence itself. Collectively, this work provided three useful tools for T. vaginalis research, advancing the potential for genetic manipulation in this protist. The factors involved in control of two important molecular events of gene expression in T. vaginalis, i.e. translation and capping were also investigated. The conclusions shed light on the processes of gene expression in this human pathogen.