Simones-Barbosa, AugustoMohammadi, Khatima2023-11-022023-11-022023https://hdl.handle.net/2292/66366Spliceosomal introns, which are distinctive features of eukaryotic genomes, are noncoding sequences excised from pre-mRNAs by the ribonucleoprotein complex spliceosome, which contribute to genome evolution and protein diversity. Spliceosomal introns and protein/RNA components have been characterised in several eukaryotic lineages; however, their origin and evolution remain unresolved. Trichomonas vaginalis (T. vaginalis) is an organism of interest in studying eukaryotic evolution, as it has a genome size of ~60MB, rich gene repertoire among protists but very few spliceosomal introns. Studies by our group have uncovered a new family of short introns in T. vaginalis. These introns feature distinctive sequences and a strict length limitation in contrast to the long introns previously described in this organism. Another study suggested that hairpin stem loops might compensate for the length of the long introns in deep-branching protists, including T. vaginalis. This is aligned with the previous observation of ‘split genes’ in the parasite Giardia lamblia joined by transsplicing, guided by intermolecular interactions between split introns. With this knowledge, I will follow up on the study of short introns in T. vaginalis by creating variants of the short introns inserted into a reporter gene. In this study, we utilised PCR, Sanger-Sequencing, transfection, and flow cytometry to answer the following questions: (i) How different are short vs. long introns in their splicing signals? (ii) How short can a T. vaginalis intron be, and could an intramolecular loop compensate for the increase in intron length? (iii) Can T. vaginalis trans-splice? In this study, we discovered that short (type B) and long (type A) introns differ in a few but significant features. These include the 5’ splice site (5’SS) motif, which is shorter or more degenerated than long introns, although it cannot tolerate a 5’-GC dinucleotide. Branch point (BP) consensus sequences, which in short and long introns are contrastingly different from each other but have one precise nucleotide. This study discovered that T. vaginalis short introns are more flexible than previously believed, provided the first evidence of trans-splicing in T. vaginalis, and showed that the intramolecular loops do not compensate for the length increase. This study furthers our understanding of spliceosomal introns in T. vaginalis and other deep-branching eukaryotes. The discoveries discussed in this thesis expand our knowledge of intron biology and eukaryotic evolution, providing new opportunities for further research in this area.Items in ResearchSpace are protected by copyright, with all rights reserved, unless otherwise indicated.https://researchspace.auckland.ac.nz/docs/uoa-docs/rights.htmhttps://creativecommons.org/licenses/by-nc-sa/3.0/nz/Thesis2023-10-31Copyright: the authorhttp://purl.org/eprint/accessRights/OpenAccess