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The evolution of RNA processing in reduced eukaryotes Grisdale, Cameron James


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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