Conference Agenda

Approximately 2/3 of the Trichomonas vaginalis genome is composed of Type II transposable elements (TEs). TEs represent a strong mutational force shaping genomic evolution, raising questions as totheir impact on T. vaginalis function and evolution. We generated the first chromosome-grade assemblies, annotation and transcriptomes offour T. vaginalis strains and three close relatives in birds: Trichmonas stableri, and two T. vaginalis-like species even more closely related to T. vaginalis. We found that the genome of T. vaginalis is massively expanded, follows a mosaic pattern of synteny, and that TEs have expanded and altered the genomic architecture. Comparison of regulatory mechanisms revealed a bias towards piRNA machinery in T. vaginalis and the least expression of TEs among all the species. We inferred the functional consequences of this TE expansion by genotyping 5,759 TE polymorphisms in 12 T. vaginalis strains and associating TE insertion with gene expression. We found 69 eQTLs (loci that explain variation in mRNA expression) in low frequency, indicating TEs likely play a deleterious role in the capacity of the parasite to regulate gene expression. This is the first genome-wide study in Trichomonas to show the evolution of TEs, their regulatory mechanisms, and their impact on transcriptional regulation.

Abstract: Spliceosomal introns, distinctive features of eukaryotic genomes, are non-coding sequences excised from pre-mRNAs by the spliceosome, contributing to genome evolution and protein diversity. Although spliceosomal introns have been characterised in several eukaryotic lineages, their origin and evolution remain unresolved. The protozoan parasite Trichomonas vaginalis is a deep-branching eukaryote with a large genome and a rich gene repertoire despite apparently having very few spliceosomal introns. We have recently uncovered a group of short introns in T. vaginalis. Here, using a reporter gene disrupted by short intron sequences, we employed an extensive series of mutagenesis to investigate their conserved features of splice signal sequences and intron length, comparing them to long introns. We found that short introns have reasonable but limited flexibility in their length, including extremely close juxtaposition of the branch and the 3’ splice sites. Additionally, their exhibit distinctive consensus sequences with a very degenerate 5’ splice site. Lastly, and surprisingly, we found that T. vaginalis is capable of trans-splicing split introns, as previously documented in Giardia lamblia, another deep-branching protozoan of the group Excavata. In conclusion, our findings expand on the diversity of introns in deep-branching eukaryotes, offering new insights into the evolution of spliceosomal introns.

Alternative splicing, a eukaryotic-originated process, involves the processing of cis-acting elements (spliceosomal introns and exons) within pre-mRNA transcripts by trans-acting elements (spliceosomal proteins and splicing factors), resulting in the generation of multiple mRNA isoforms. Alternative splicing is tightly linked to eukaryotic complexity. Spliceosomal introns have expanded despite consistent spliceosomal protein composition from yeast to humans. This raises questions about the emergence of the spliceosome during eukaryotic evolution and the proteins influencing the complexity of spliceosomal introns. Considering that protists evolved over 500 million years before yeast, studying alternative splicing in Giardia duodenalis, an early eukaryotic protist, could provide insights into the origins of the spliceosome and spliceosomal introns. Cis- and trans-factors guiding alternative splicing in Giardia are understudied. We conducted a comprehensive bioinformatic analysis of annotated intronic and proteomic data from the basal protist Giardia to complex protist Plasmodium falciparum, alongside other model eukaryotes (Arabidopsis thaliana, Caenorhabditis elegans, Drosophila melanogaster, Mus musculus, and Saccharomyces cerevisiae). Our preliminary ortholog mining suggests the presence of spliceosomal proteins across established spliceosomal complex sub-categories in Giardia. This study represents one of the first attempts to comprehend the spliceosome and spliceosomal introns at the base of eukaryogenesis, shedding light on the origin of alternative splicing.

The central dogma (DNA-to-RNA-to-Protein) is key to cellular regulation, with chromatin/transcriptional and post-translational tiers achieving complexity in higher eukaryotes. Post-transcriptional regulation (PTR) by RNA-binding proteins (RBPs) is ancient and ubiquitous, expanding significantly with eukaryotic evolution, making it central to cellular life. We investigated whether novel RBPs emerged during the prokaryotic-to-eukaryotic transition. Our phylogenomic atlas across the tree of life reveals that the eukaryotic RBPome is shaped by bacterial and archaeal RBP systems, alongside the emergence of “novel” RBP families. In the early-branching eukaryote Giardia duodenalis, predating yeast by a billion years, RBPs for RNA-splicing, RNA-silencing, translation repression, and cellular fate regulation emerged. We characterized the G. duodenalis RBPome through in silico modelling, transcriptomic, proteomic profiling, and interactome capture, revealing canonical and non-canonical RBPs. Functional genetics, RNA-network capture, and phase-separation assays of key eukaryotic originated RBPs, including early Pumilio homologs (PUF, PUM) and helicases DDX3x and EIF4A, and other non-canonical RBPs like Phospho-glycerate kinase suggest Giardia RBPs exhibit complexity similar to higher eukaryotes, with roles in translational repression, biomolecular condensates, and cell differentiation. Our findings indicate that complex RBP regulation emerged early in eukaryotic evolution, potentially pivotal for the emergence and evolution of eukaryotes.