Naomi Fast

Associate Professor

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Dissertations completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest dissertations.

Spliceosomal evolution in the reduced genomes of microsporidia (2023)

Eukaryotic genes are mosaics of coding regions and non-coding regions called spliceosomal introns. Introns are removed from pre-mRNA by a ribonucleoprotein complex called the spliceosome. The spliceosome in humans is composed of five small nuclear RNAs and nearly 200 associated proteins. As nuclear genomes reduce, introns are more readily lost and, as a result, there is weakened selection to maintain a complex spliceosome. My dissertation focuses on the evolution of introns and spliceosomes in species with reduced genomes. Microsporidia are intracellular parasites that have extremely reduced nuclear genomes and have lost of many, if not all, introns. I examined all available microsporidian genomes and identified a family of introns that are conserved in all intron-containing microsporidia. These introns differ from typical microsporidian introns: they are longer, are spliced at higher levels, have hyperextended motifs, and contain internal conserved regions that may indicate a non-coding function. Despite the genome of Pseudoloma neurophilia having no annotated introns, I identified the presence of two long introns. I established that the P. neurophilia spliceosome is the most reduced known, and that the retained proteins and small nuclear RNAs are notably divergent from canonical orthologs. Due to extensive sequence evolution in microsporidia, direct comparisons to model organisms can be difficult. I produced the first transcriptomes from the Rozellomycota, the sister group to microsporidia, and found significant plasticity of expression throughout the lifecycle. I also found that Rozella introns are extremely constrained in size but lack conserved spliceosomal motifs. These data, combined with available genomic data from two other related parasites, provide insights into the evolution of microsporidian introns. Spliceosomal reduction extends beyond the loss of proteins. In the red alga Cyanidioschyzon merolae, a regulatory toggle in the core spliceosomal protein Prp8 has been lost. I examined a diverse set of eukaryotes and found that convergent losses occurred in other reduced taxa, including microsporidia. Overall, my dissertation highlights the usefulness of microsporidia as a model for spliceosomal reduction and aids in our understanding of the evolution of splicing in reduced systems.

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Insights into the evolution of pre-mRNA splicing from reduced algal genomes (2022)

Eukaryotic genes are interrupted by non-coding regions known as introns, which are removed through pre-mRNA splicing. This process catalyzed by the spliceosome, a large complex of five small nuclear ribonucleoproteins (snRNPs), and various other proteinaceous accessory complexes. Although this process is highly conserved across eukaryotes, significant divergence is seen in organisms with reduced genomes. My dissertation research explores the evolution of nuclear pre-mRNA splicing and the spliceosome in diverse reduced algal genomes. I examined transcriptomes of the cryptophyte Guillardia theta and the chlorarachniophyte Bigelowiella natans for splicing in their nucleomorphs, the remnant nuclei found in their plastids. There, I observed high levels of unspliced transcripts in the intron-sparse G. theta nucleomorphs, and typical eukaryotic levels of splicing in the many tiny nucleomorph introns of B. natans. To determine if this reflects contrasting intron richness and/or differing ancestries of these plastids, I investigated splicing in three species of red algae: the mesophilic Porphyridium purpureum, and the extremophilic Cyanidiales red algae Cyanidioschyzon merolae and Galdieria sulphuraria. A comparison of transcriptome data revealed extremely low levels of splicing in the intron-poor C. merolae, typical splicing in the intron-rich G. sulphuraria, and an intermediate level of splicing in P. purpureum, whose intron richness lies between C. merolae and G. sulphuraria. My analyses present a trend where transcripts more often remain unspliced in genomes with low intron numbers, and spliceosomes exhibit increasing divergence and loss of components. I also showed that an intron’s splicing level is tied to 5′ splice site and branch donor sequence variability, suggesting an increased reliance on base-pairing for intron recognition in an increasingly reduced spliceosome. I explored potential functions of persisting nucleomorph introns of the cryptophytes Guillardia theta and Chroomonas mesostigmatica. I performed differential analysis of light and dark transcriptomes for both, and assembled a nuclear transcriptome for C. mesostigmatica, showing extensive intron retention in cryptophyte nucleomorph transcripts. However, differential splicing and gene expression is limited in nucleomorphs, suggesting that secondary plastids are primarily regulated by nuclear genes. My research highlights patterns of divergence in splicing in genomes evolving under the pressures of reduction.

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Microsporidian Diversity of the Pacific Northwest: Microsporidiosis in a Marine Nematode (2015)

Microsporidia are a diverse group of spore-forming obligate endoparasitic fungi that have over 1,300 named species. Microsporidian spores are ubiquitous in the environment, but species diversity remains vastly unexplored, as the total number of species is suspected to equal that of their hosts. Microsporidian parasites are also model organisms for the study of genome reduction since they possess some of the smallest eukaryotic genomes known to date.To examine the diversity of microsporidian parasites in the environment, I screened for the presence of microsporidian parasites in poorly sampled reservoirs: soil, sand, and compost. I amplified ribosomal DNA (rDNA) sequences for 23 undescribed and three described microsporidian species that were highly diverse phylogenetically, including representatives from four of the five major microsporidian clades. Molecular screening for the hosts revealed that one undescribed microsporidian infected a free-living marine nematode (Odontophora rectangula). I characterized the infection and ultrastructure of the parasite by transmission electron microscopy and fluorescent in situ hybridization. It is a novel microsporidian that I named Sporanauta perivermis (“marine spore of roundworms”). S. perivermis infects the hypodermal, muscle, and reproductive tissues of adult O. rectangula. However, the infection pattern differed between genders where only reproductive tissues were infected in adult females (uteri and eggs), suggesting that S. perivermis is transmitted vertically. Juvenile hosts showed similar infection patterns to adults, and infection pattern allowed prediction of host gender prior to adulthood.Phylogenetic analyses revealed that S. perivermis is sister to a clade containing the Daphnia-infecting microsporidian Ordospora colligata and the Encephalitozoon lineage (five species), which contains three species that infect humans. A genomic survey of S. perivermis indicated high levels of similarity in gene content (over 90%), gene length, and synteny between S. perivermis, O. colligata and Encephalitozoon. S. perivermis and O. colligata shared chromosomal arrangements that were not present in Encephalitozoon genomes, including chromosomal rearrangements that could be linked to genome reduction mechanisms. The genome size of S. perivermis is at least 2.2 Mbp, but is likely larger since its intergenic regions were longer, on average, than those of relatives with genome sizes of 2.3-3.0 Mbp.

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The Evolution of RNA Processing in Reduced Eukaryotes (2014)

RNA-processing encompasses several critical steps in the regulation of gene expression. Both transcription and pre-mRNA splicing are important for the formation of mature RNA. Most eukaryotic genes are interrupted by introns, the removal of which is catalyzed by the spliceosome. The spliceosome is a large molecular machine comprised of five small nuclear RNAs (snRNAs) and up to two hundred proteins. In addition to constitutive removal of introns, alternative splicing increases transcriptome complexity, as it allows for the formation of multiple transcript isoforms from a single pre-mRNA. Although these processes are well-studied in model systems, relatively little is known about their evolution in unicellular eukaryotes. To investigate RNA-processing in reduced systems, I examined the transcriptomes of the microsporidian parasite Encephalitozoon cuniculi, and the red alga Cyanidioschyzon merolae. E. cuniculi and C. merolae harbour reduced genomes of 2.9Mbp and 16.5Mbp, respectively. Both genomes were annotated with fewer than 30 spliceosomal introns, and both have undergone reduction in spliceosomal components, including the loss of the U1 snRNA. Illumina RNAseq was used to sequence the transcriptomes of E. cuniculi at three time-points during its intracellular stage, and C. merolae under light and dark phases of its growth cycle. I found extremely low levels of pre-mRNA splicing for nearly all intron-containing genes in both organisms, under all conditions examined. These levels of splicing appear to be lower than in any other eukaryote examined, suggesting that reduction in unrelated spliceosomes reveals a common evolutionary trend: decreased splicing efficiency.In addition to intron-retention, I found examples of other types of alternative splicing in these two reduced systems. C. merolae displayed all major types of alternative splicing, and some events occurred at relatively high frequencies. The presence of few or no alternative splicing regulatory protein-coding genes in C. merolae and E. cuniculi, respectively, made this finding especially surprising. Also, I found high levels of antisense transcription in C. merolae, with the potential to play a regulatory role in gene expression.

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Evolution of the Microsporidian Genome and Gene Expression (2009)

No abstract available.

 

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