Transcriptome analysis of the parasite Encephalitozoon cuniculi: an in-depth examination of pre-mRNA splicing in a reduced eukaryote

Abstract

Background: The microsporidian Encephalitozoon cuniculi possesses one of the most reduced and compacted eukaryotic genomes. Reduction in this intracellular parasite has affected major cellular machinery, including the loss of over fifty core spliceosomal components compared to S. cerevisiae. To identify expression changes throughout the parasite's life cycle and also to assess splicing in the context of this reduced system, we examined the transcriptome of E. cuniculi using Illumina RNA-seq.

Results: We observed that nearly all genes are expressed at three post-infection time-points examined. A large fraction of genes are differentially expressed between the first and second (37.7%) and first and third (43.8%) time-points, while only four genes are differentially expressed between the latter two. Levels of intron splicing are very low, with 81% of junctions spliced at levels below 50%. This is dramatically lower than splicing levels found in two other fungal species examined. We also describe the first case of alternative splicing in a microsporidian, an unexpected complexity given the reduction in spliceosomal components.

Conclusions: Low levels of splicing observed are likely the result of an inefficient spliceosome; however, at least in one case, splicing appears to be playing a functional role. Although several RNA decay genes are encoded in E. cuniculi, the lack of a few key players could be reducing decay levels and therefore increasing the proportion of unspliced transcripts. Significant proportions of genes are differentially expressed in the first forty-eight hours but not after, indicative of genetic changes that precede the intracellular to infective stage transition.

Figure 1

Differential expression across three post-infection time-points. Plot of log2 fold change versus mean expression level for all E. cuniculi genes. Red dots indicate those genes that are differentially expressed and black dots indicate those that are not. (A) Differential expression between 24 hr and 48 hr, (B) 24 hr and 72 hr, and (C) 48 hr and 72 hr post-infection.

Figure 2

Splicing levels of all E. cuniculi intron-containing genes. Levels of splicing were determined by measuring the number of spliced and unspliced transcripts and dividing spliced by total transcripts to produce a percentage of splicing. From left to right, splicing levels at 24 hr, 48 hr, and 72 hr are indicated by grey bars. E. cuniculi gene names are on the x-axis. Significant differences in splicing levels between time-points are shown by darkened boxes along the x-axis. From left to right, darkened boxes indicate significant differences between 24 hr and 48 hr, 24 hr and 72 hr, and 48 hr and 72 hr.