Report Microsporidia Evolved from Ancestral Sexual Fungi

Summary Microsporidia are obligate, intracellular eukaryotic pathogens that infect animal cells, including human

miscRNAs in <i>C. neoformans</i> var. <i>grubii</i>.

<p><b>A.</b> Two examples of a miscRNA as visualized through Artemis. The coverage of the plus stand is represented by the black curve. The coverage of the minus strand is represented by the blue curve. These results were obtained when cells grown in low glucose and nitrogen medium (starvation medium) underwent strand-specific sequencing. F1, F2, and F3 stand for 5′ to 3′ frames 1, 2, and 3, respectively. F4, F5, and F6 stand for 3′ to 5′ frames 1, 2 and 3, respectively. The small black vertical bars indicate the position of the stop codons for each frame. <b>B.</b> Schematic representation of the positions of the miscRNAs in the <i>C. neoformans</i> var. <i>grubii</i> genome as compared to coding sequences. The numbers of miscRNAs at each position is indicated. The number of miscRNAs in the antisense strand of other miscRNAs is indicated between brackets.</p

Antisense/sense transcription in <i>C. neoformans</i> var. <i>grubii</i>.

<p><b>A.</b> Comparison of sense/antisense transcription when an antisense transcript is present. Strand-specific data obtained from cells grown on YPD is shown. The BaseMean values represent the normalized reads count for each transcript and measure the level of sense transcription (x axis) and antisense transcription (y axis) as calculated by DESeq <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004261#pgen.1004261-Anders1" target="_blank">[126]</a>. Outliers with a BaseMean above 12,000 were not represented. <b>B.</b> Example of differential expression of miscRNA antisense of a coding gene as observed through Artemis. The red curve represents the non-strand-specific coverage observed when cells were grown in YPD to stationary phase at 30°C (condition 1); the green curve shows the non-strand-specific coverage observed when the cells were grown in YPD to log phase at 30°C (condition 2). F1, F2, and F3 stand for 5′ to 3′ frames 1, 2, and 3, respectively. F4, F5, and F6 stand for 3′ to 5′ frames 1, 2, and 3, respectively. The small black vertical bars indicate the position of the stop codons for each frame. <b>C.</b> Northern blot obtained after hybridization with strand-specific probes. RNA was extracted from cells growing in YPD (2×10⁸ cells/mL) at 30°C (condition 1), YPD (5×10⁷ cells/mL) at 30°C (condition 2), YPD with 0.01% SDS (5×10⁷ cells/mL) at 30°C (condition 3), YPD with 10 mg/mL fluconazole (5×10⁷ cells/mL) at 30°C (condition 4), YPD (5×10⁷ cells/mL) at 37°C (condition 5), and YP galactose (2×10⁸ cells/mL) at 30°C (condition 6) in duplicate. Then, 5 µg RNA were loaded on a denaturing electrophoresis agarose gel, electrophoresed, and transferred to a nylon membrane. The horizontal black line represents the position of the probes.</p

Identification of replication origins.

<p>The two sets of three panels show 2D gel patterns of replication intermediates in the regions of CnORI1.168 and CnORI1.228, as diagramed below. The arcs of bubble-shaped replication intermediates, Y-shaped replication intermediates, and replication intermediates are labeled on the upper left panel. The upper autoradiogram in each set shows the 2D gels of the <i>Eco</i>RI fragments that defined the origins. The two lower autoradiograms in each set show 2D gels of genomic restriction fragments that overlap the <i>Eco</i>RI fragments, also shown in the diagrams below. CnORI1.228: upper panel, 4,722-bp <i>Eco</i>RI fragment; lower left panel, 4,655-bp <i>Xho</i>I fragment overlapping left end of <i>Eco</i>RI fragment; lower right panel, 6,297-bp <i>Sca</i>I fragment overlapping right end of EcoRI fragment. CnORI1.168: Upper panel, 5,728-bp <i>Eco</i>RI fragment; lower left panel, 4,803-bp <i>Xho</i>I-<i>Nhe</i>I fragment overlapping left end of <i>Eco</i>RI fragment; lower right panel, 4,810-bp <i>Cla</i>I-<i>Sac</i>II fragment overlapping right end of <i>Eco</i>RI fragment. See text for details.</p

Polyadenylation sites in <i>C. neoformans</i> var. <i>grubii</i>.

<p><b>A.</b> Poly(A) reads are enriched within 500 nt from the stop codon of the gene models. <b>B.</b> Examples of CR-APA and UTR-APA as visualized through Artemis. The black arrows indicate the position of the alternative poly(A) sites. The green curves indicate the plus strand coverage. The small black vertical bars indicate the position of the stop codons for each frame. <b>C.</b> Sequence composition around the poly(A) cleavage sites. <b>D.</b> Proportion of the variants of the AUGHAH motif. <b>E.</b> Relative position of the poly(A) site (nt) to the AUGHAH motif in coding genes and miscRNAs. <b>F.</b> Efficiency of the poly(A) sites to induce transcription termination according to the presence or absence of an AUGHAH motif.</p

Alternative splicing in <i>C. neoformans</i> var. <i>grubii</i>.

<p><b>A.</b> Examples of alternative splicing. F4, F5, and F6 stand for 3′ to 5′ frames 1, 2 and 3, respectively. The small black vertical bars indicate the position of the stop codons for each frame. The numbers for each type of alternative splicing events annotated in the genome are indicated between brackets. <b>B.</b> Evaluation of intron retention level in <i>C. neoformans</i> according to the ratio of transcription intron/exon threshold used is represented.</p

Differentially expressed gene clusters.

<p>Genes differentially expressed between the three conditions (PG, pigeon guano; SM, starvation medium; YPD, rich media) were identified from strand-specific RNA-Seq using EdgeR with two biological replicates per condition (rep1, rep2). Expression profiles are ordered based on hierarchical clustering tree; 6 clusters were defined using the kmeans algorithm (Material and Methods).</p

Organization of the centromeres in <i>C. neoformans</i> strain H99 and a comparison with other serotypes.

<p><b>A.</b> Schematic showing the distribution of transposons, Tcn1–Tcn6, in the presumptive centromeres of all 14 chromosomes of <i>C. neoformans</i> strain H99. Each region was identified as the largest ORF-free region on its respective chromosome and contains transposons or its footprints, which are clustered in these sites. <b>B.</b> A comparative analysis of the largest ORF-free regions predicted to be centromeres between <i>C. neoformans</i> var. <i>grubii</i> (H99), <i>C. neoformans</i> var. <i>neoformans</i> (JEC21 and B3501A), and <i>C. gattii</i> (WM276 and R265) using FungiDB reveal conserved synteny of the flanking genes in chromosome 14. The grey color represents the regions that show synteny among different strains. The ORFs present in the centromeric regions are either pseudogenes or have similarity with transposons. <b>C.</b> ChIP-Seq analysis showed the enrichment of a conserved kinetochore protein, CENP-C, at the centromeric regions. Here, the enrichment on centromeric region of chromosome 14 (<i>CEN14</i>) is shown. The upper panel shows the enrichment on the whole chromosome. In the lower panel, the putative centromeric region is enlarged to show the enrichment profile of CENP-C. <b>D.</b> RNA-Seq analysis reveals the absence of poly(A) RNA from <i>CEN14</i>. <b>E.</b> Targeted truncation mutagenesis on either side of the <i>CEN14</i> centromere DNA. Four DNA fragments were produced and transformed into a diploid strain of <i>C. neoformans</i>. The stick-and-ball represents the telomeric seed sequence added to the constructs by amplification with primer GI003. No targeted recombination was observed for two constructs, whereas the other two PCR analyses indicated integration of the DNA in those regions. <b>F.</b> PCR confirmation of recombination. Lanes 1–3 contain PCRs with primers ai270-GI033, and lanes 5–7 contain PCRs with primers ai270-GI034. Lanes 1 and 5 are amplification results from the diploid strain AI187; lanes 2 and 6 are from strains with integration on the left and right sides, respectively; and lanes 3 and 7 are negative PCR controls. Lane 4 is the Invitrogen 1 kb+ size marker.</p