Fig 1.
Evolutionary analysis of Saccharomyces genes involved in RNA metabolism.
(A) Schematic of the lifecycle of the L-A virus of S. cerevisiae. Starting at the bottom of the figure, new viral positive sense single-stranded RNA (+ssRNA) is synthesized within the L-A virus capsid and extruded into the cytoplasm. The enzymatic activity of the viral Gag protein “steals” cap structures from host mRNAs and conjugates them to viral +ssRNAs. The capped viral +ssRNA is used as a template for translation and any remaining uncapped +ssRNA is encapsidated to form new viral particles by interaction with the L-A polymerase protein. Packaged +ssRNA is used as a template during negative strand synthesis to produce viral genomic dsRNA. (B) A cartoon representation of 5’-to-3’ and 3’-to-5’ RNA degradation by Xrn1p and the SKI/exosome complex, respectively, adapted from Parker et al. [29]. (C) Evolutionary analysis of XRN1 and genes of the SKI/exosome complex. An alignment of each gene was analyzed using 4 common tests for positive selection (PAML, FEL, REL, and MEME) as fully described in S1 Table. “Yes” indicates that there is positive selection detected in this gene by the indicated test.
Fig 2.
The effect of XRN1 evolution on the restriction of the L-A and killer viruses of S. cerevisiae.
(A) (Top row) dsRNA extraction from S. cerevisiae xrn1Δ with or without complementation by XRN1 from different species of Saccharomyces (S. cer—S. cerevisiae; S. mik—S. mikatae; S. kud—S. kudriavzevii; S. bay—S. bayanus). From the same agarose gel, the first two lanes were spliced to the last three lanes for clarity. (Bottom two rows) Western blots showing the expression of HA-tagged Xrn1p and Adh1p (control) within each complemented strain. (B) (Top) Domain diagrams of Xrn1p showing the position of the catalytic residue E176 and the location of the C-terminal truncations of Xrn1p (dashed line). Xrn1p with the E176G mutation or the Δ1206–1528 truncation are catalytically inactive, as described previously [49]. (Bottom) dsRNA extraction from S. cerevisiae xrn1Δ expressing wild type XRN1, the catalytically inactive XRN1(E176G) or truncation mutants of Xrn1p (all derived from S. cerevisiae). (C) A representative picture of S. cerevisiae killer (L-A+ Killer+) and non-killer (L-A- Killer-) yeasts and the effect of killer toxin expression on the growth of a lawn of sensitive yeast. (D) The effect of Xrn1p expression on the diameter of the kill zones around killer yeast. Asterisks are indicative of a significant difference in mean kill zone area compared to all other samples tested (Tukey-Kramer test p<0.05). See S2 Fig for representative images of the kill zones.
Fig 3.
XRN1 orthologs vary in their ability to cure S. cerevisiae of the L-A and killer viruses.
(A) Killer S. cerevisiae strains over-expressing Xrn1p orthologs (with or without C-terminal HA tag) were assayed for loss of the killer phenotype resulting in “cured” clones. The percentage of cured clones is indicated. (B) Representative data from 16 clonal isolates either not expressing (left) or expressing XRN1 from S. cerevisiae (right), with the lack of kill zone indicating a cured clone. (C) Non-quantitative RT-PCR was used to confirm the loss of L-A and killer RNAs from strains deemed to have lost the killer phenotype from Fig 3A and 3B, compared to the parental uncured strain (L-A+ Killer+) and S. cerevisiae BY4741 (L-A+ Killer-).
Fig 4.
Xrn1p housekeeping functions have been conserved during Saccharomyces speciation.
(A) The doubling time of S. cerevisiae xrn1Δ with and without complementation with XRN1 from different species of Saccharomyces (error bars represent SEM, n = 3). Below are representative pictures of the growth and morphology of individual colonies. (B) The growth and benomyl sensitivity of S. cerevisiae xrn1Δ cultured on YPD solid medium, and the effect of complementation with XRN1 from different species of Saccharomyces. (C) The effect of over-expression of each XRN1 ortholog on the growth of S. cerevisiae upon solid medium, compared to the over-expression of GFP. Cells are grown on medium containing either raffinose or galactose as the sole carbon source to control the activity of the GAL1 promoter. (D) Ty1 retrotransposition within S. cerevisiae xrn1Δ complemented by XRN1 from different species, relative to XRN1 from S. cerevisiae. All error bars represent SEM, n>3.
Fig 5.
A structured protein domain within Xrn1p is responsible for species-specific antiviral activity.
(A) Space-filled structural model of S. cerevisiae Xrn1p generated by Phyre analysis [58]. The D1 domain is colored orange. Amino acids 354–503, 979–1109, and 1240–1528 are unresolved in the model due to a lack of structural information. The structural model is included as S1 File. (B) A representative amino acid alignment shows two variable sites within the Xrn1p D1 domain that are predicted to be evolving under positive selection. (C) A linear diagram of S. cerevisiae Xrn1p based on the structure of K. lactis Xrn1p, indicating select domains of the C-terminus and showing the conservation of Xrn1p across Saccharomyces species. The white domain has an unknown structure and is predicted to be unstructured by Phyre analysis [58]. Triangles indicate the position of sites deemed to be under positive selection by four site-based models of molecular evolution, PAML M8 (red), REL (black), FEL (white), and MEME (green). See S1 Table for a list of all sites. The seven residues that form the catalytic pocket of Xrn1p are shown as vertical dotted lines within the N-terminal domain. (D) (Left) Chimeric proteins derived from the fusion of domains of Xrn1p from S. cerevisiae (white) and S. kudriavzevii (black). Relevant domains within Xrn1p are colored according to Chang et al. (D1 (orange) 731–914; D2 (green), 915–960 and 1134–1151; D3 (magenta); 978–1108) [59]. (Right) Clonal isolates of a killer S. cerevisiae strain expressing chimeric Xrn1p were assayed for their ability to cure the killer phenotype from S. cerevisiae (error bars represent SEM, n>3).
Fig 6.
The totivirus structural protein Gag is associated with Xrn1p in vivo.
Western blot analysis of Xrn1p and L-A Gag co-immunoprecipitation. (A) (Top) V5-tagged and untagged Gag proteins were immunoprecipitated in the presence of Xrn1p-HA from either S. cerevisiae or S. kudriavzevii. (Bottom) HA-tagged and untagged Xrn1p from either S. cerevisiae or S. kudriavzevii were immunoprecipitated in the presence of Gag-V5. (B) Native, untagged, virus encoded L-A Gag was immunoprecipitated in the presence of Xrn1p-HA from either S. cerevisiae or S. kudriavzevii using beads with (+Ig) or without (-Ig) anti-Gag antibody present. Adh1p was used in all panels as a loading control to ensure equal input of total protein and the specificity of immunoprecipitation.
Fig 7.
The description of a novel totivirus from S. kudriavzevii and its unique sensitivity to restriction by Xrn1p from its cognate host species.
(A) dsRNA extraction from different species of Saccharomyces yeasts including S. cerevisiae (BY4741), S. mikatae (JRY9181), S. kudriavzevii (FM1183), and S. bayanus (JRY8153). The major product of the extraction method is a single ~4.6 kbp dsRNA species, as shown by agarose gel electrophoresis. (B) A schematic representation of the genome organization of the totivirus SkV-L-A1 from S. kudriavzevii. H154 represents the conserved catalytic histidine used for totivirus cap-snatching [14]. (C) The evolutionary relationship of SkV-L-A1 to known totiviruses was inferred by using the Maximum Likelihood method with bootstrap values from 100 replicates shown at each node. The nucleotide sequence of the POL gene was aligned from six totiviruses (GenBank accession numbers: SkV-L-A1 (this study; KX601068), L-A-lus (JN819511), L-A (NC_003745), tuber aestivum virus 1 (TAV1) (HQ158596), black raspberry virus F (BRVF) (NC_009890), L-BC (NC_001641)). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The exact phylogenetic relationship of the “L-A-like” viruses is somewhat ambiguous due to low bootstrap support within the clade (S6 Fig). (D) S. kudriavzevii was used to express HA-tagged XRN1 from different Saccharomyces yeasts (S. cer—S. cerevisiae; S. mik—S. mikatae; S. kud—S. kudriavzevii; S. bay—S. bayanus), with their expression measured by Western blotting, relative to the expression of ADH1. (E) Relative abundance of SkV-L-A1 RNAs when XRN1 from different Saccharomyces species are over-expressed within S. kudriavzevii, as determined by RT-qPCR. The asterisk represents a p-value of <0.05 (Tukey-Kramer test).