sRNAs as possible regulators of retrotransposon activity in Cryptococcus gattii VGII

Abstract

Background: The absence of Argonaute genes in the fungal pathogen Cryptococcus gattii R265 and other VGII strains indicates that yeasts of this genotype cannot have a functional RNAi pathway, an evolutionarily conserved gene silencing mechanism performed by small RNAs. The success of the R265 strain as a pathogen that caused the Pacific Northwest and Vancouver Island outbreaks may imply that RNAi machinery loss could be beneficial under certain circumstances during evolution. As a result, a hypermutant phenotype would be created with high rates of genome retrotransposition, for instance. This study therefore aimed to evaluate in silicio the effect of retrotransposons and their control mechanisms by small RNAs on genomic stability and synteny loss of C. gattii R265 through retrotransposons sequence comparison and orthology analysis with other 16 C. gattii genomic sequences available.

Results: Retrotransposon mining identified a higher sequence count to VGI genotype compared to VGII, VGIII, and VGIV. However, despite the lower retrotransposon number, VGII exhibited increased synteny loss and genome rearrangement events. RNA-Seq analysis indicated highly expressed retrotransposons as well as sRNA production.

Conclusions: Genome rearrangement and synteny loss may suggest a greater retrotransposon mobilization caused by RNAi pathway absence, but the effective presence of sRNAs that matches retrotransposon sequences means that an alternative retrotransposon silencing mechanism could be active in genomic integrity maintenance of C. gattii VGII strains.

Keywords: Cryptococcus gattii; RNAi; Retrotransposons; Synteny.

Fig. 1

Retrotransposon number, density and genome size comparison. The red line indicates the number of identified retrotransposons in each strain; blue line represents retrotransposon density in C. gattii genomes. The green line shows the size of C. gattii assembled genomes

Fig. 2

Orthologous sequences count. Orthologous sequences from C. gattii WM276 and R265 shared with VGI, VGII, VGIII, and VGIV strains

Fig. 3

Sequence count of orthologous and syntenic retrotransposons. Bars indicate the total amount of orthologous and syntenic sequences shared by VGII (a) and VGI (b) strains with R265 and WM276 genomes, respectively

Fig. 4

Phylogenetic tree for reverse transcriptase (RT) and RNase H domains. Domains of reverse transcriptase (a) and ribonuclease H (b) from C. gattii retrotransposons to orthologous R265.19 sequence. Sequences that were direction-adjusted by MAFFT aligner are indicated by the “R” before strain name. Model inference was performed by jModelTest and phylogeny reconstruction by Mr. Bayes using the Kimura-2-parameters substitution model

Fig. 5

mRNA and sRNA retrotransposon correlation. RPKM values of C. gattii R265 retrotransposons for mRNA and sRNA expression of wild-type (WT) C. gattii R265 in a zinc deprivation culture

Fig. 6

mRNA and sRNA reads profile mapping. Distribution of aligned reads from a mRNA and b sRNA libraries in terminal repeats, protein domains, and spacer regions inside retrotransposon sequences. Abbreviations: Long Terminal Repeats (LTR), Integrase (INT), Ribonuclease H (RNAse), Reverse Transcriptase (RT), Aspartyl Protease (PROT)