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

Abstract

BackgroundThe 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.ResultsWe 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.ConclusionsLow 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.

The Genome of Spraguea lophii and the Basis of Host-Microsporidian Interactions

“…Of the eight putative intron-containing genes we identified in Spraguea , five have orthologues in E. cuniculi that also contain an intron (S17, L27a, S24, L5 and poly(A) binding protein), and the efficiency with which those introns are spliced parallels our results with the S. lophii transcriptome. The introns in E. cuniculi S17, L27a, S24 and L5, for which we did not detect splicing in Spraguea , are also short [49] and are among the least efficiently spliced genes in E. cuniculi , with less than 15% of transcripts experiencing splicing (a figure which drops to 5% for L5) [50]. In contrast, the E. cuniculi orthologue of the actively-spliced poly(A) binding protein contains the longest and most frequently spliced intron in E. cuniculi , with over 80% of transcripts spliced.…”

Transcriptome sequencing and characterization of ungerminated and germinated spores of <italic>Nosema bombycis</italic>

“…Genes encoding PTPs and SWPs in Encephalitozoon cuniculi were remarkably up-regulated from 24 to 48 h and from 24 to 72 h post-infection, respectively. These results indicated that mRNAs which were produced in higher quantity during the sporogony stage are then accumulated in the spores [10]. Our result is in agreement with this research.…”

“…High-throughput mRNA sequencing (RNASeq) offers the possibility to measure the transcript expression levels in different samples using a single assay [42][43][44]. Compared with the transcriptome analysis of Encephalitozoon cuniculi conducted in 2013 [10], these researchers obtained ∼9-17 million reads at three post-infection time-points with a genome size of 2.9 Mbp. In this study, we produced an N. bombycis reference transcriptome consisting of ∼30 million high-quality reads with a genome size of 15.7 Mbp.…”

“…Some genomes of important microsporidia have been reported and analyzed [7][8][9]. Based on the understanding of the microsporidia genome, transcriptome studies have been widely carried out [9][10][11], which generated information on the changes of total mRNA during a particular period. The genome of N. bombycis was published in 2013, and studies have shown that the genome of N. bombycis is expanded, rather than compact as some other parasitic microsporidian genomes are through several common molecular mechanisms [12].…”

Horizontally acquired genes in early-diverging pathogenic fungi enable the use of host nucleosides and nucleotides

“…TK is an especially promising target for disruption because of the apparent importance placed on dTMP synthesis during evolution and the wide range of TK-activated prodrugs currently used as antiviral therapies. First, we confirmed that TK is expressed by analyzing a previously published E. cuniculi transcriptome dataset (32); TK was in the top quartile of expressed genes at all three postinfection time points (Table S3 and Dataset S4). We then evaluated the predicted TK proteins of E. cuniculi, N. parisii, and R. allomycis in an assay in which the nucleoside kinase activity of TK would activate the prodrug 5-fluoro-2-deoxyuridine (FUdR), converting it into the toxic compound fluorodeoxyuridine monophosphate and killing the tester strain of Saccharomyces cerevisiae (31).…”