Fig 1.
ncRNAs repertoire and transcriptional evidence in Leishmania parasites.
A. Distribution of ncRNAs identified in all species. Those ncRNAs representing less than 0.5% were categorized as “Others”. The presence of a large proportion of non-categorized ncRNAs (unclassified) are elements that could not be assigned to any group due to a lack of biological information related to their secondary structure motifs or functional annotation in the original database. B. ncRNAs gene prediction was validated by transcriptional evidence using RNA-seq analysis. We observed expression for a rate between 46.3% (L. braziliensis) to 97.55% (L. major) of predicted ncRNAs. C. Comparative expression values of log2 counts per million (log2 CPM) normalized read counts. The top five representative ncRNAs according to the number of predictions are represented, together with the coding genes expressed in L. braziliensis, L. donovani, and L. major.
Fig 2.
ncRNA conservation analysis through Leishmania spp.
A. Flower map indicating the ncRNA conservation across25 Leishmania parasites. The inner circle represents the core-RNAome, while the outer circle displays the accessory-RNAome Each petal represents species-specific ncRNAs identified for each evaluated species. B. Abundance of ncRNA families in the pan-RNAome of Leishmania spp. C. Upset plot corresponding to the top 25 conserved clusters within all 25 genomes. In Purple are represented the clusters conserved exclusively in species of L. donovani complex, related to visceral leishmaniasis (VL) (L. donovani and L. infantum strains), while the clusters associated with Viannia subgenus (L. braziliensis, L. panamensis, and L. peruviana species) are in green. The core clusters are in blue. Pie charts for the Viannia subgenus and L. donovani complex lineages illustrate the relative abundance of different ncRNA classes.
Fig 3.
Phylogenetic analysis of the Leishmania genus and comparison with clustering of core RNA families presence/absence.
The phylogenetic tree on the left is based on orthologous gene identity comparisons across multiple Leishmania species and subspecies. The dendrogram on the right represents the clustering of RNA families based on the presence/absence of core RNAome elements, calculated using the Jaccard coefficient similarity matrix. Nodes are colored according to Leishmania subgroups: the Leishmania subgenus (yellow), Sauroleishmania subgenus (green), and Viannia subgenus (purple). The tree scale is indicated as 0.01 substitutions per site.
Fig 4.
Differentially expressed coding and non-coding genes through developmental stages in L. braziliensis.
A. Differentially expressed genes in amastigote, metacyclic, and procyclic developmental stages. The number on top of each bar represents all overexpressed genes in each stage comparison. The shadow bar represents the ncRNAs overexpressed. B. Venn diagram highlighting the exclusively DEGs of each developmental stage. C. Top 10 GO biological processes enriched terms (p-value) according to the exclusive genes for each developmental stage. Metacyclic stage is left empty to indicate that have not enrichment processes.
Fig 5.
Co-expression module-developmental stages associations for L. braziliensis.
Each row corresponds to a module and the columns to a developmental stage. Each cell contains the corresponding correlation and p-value (in parenthesis). The table is color-coded by correlation according to the color legend. The heatmap shows that modules M4 (R2 = 098 and p < 0.001), M5 (R2 = 0.79 and p < 0.001) have the major correlation with the procyclic developmental stage, besides M2 (R2 = 0.86 and p < 0.001), M3 (R2 = 0.66 and p < 0.001) are tightly related to the metacyclic stage and modules M1 (R2 = 0.92 and p < 0.001); and M9 (R2 = 0.67 and P < 0.001) are correlated to the amastigote developmental stage.
Fig 6.
Biological processes GO enrichment of hub ncRNAs related to amastigote and procyclic promastigote developmental stages in L. braziliensis.
A and B. Sub-networks of the hub genes related to amastigotes (A) and procyclic promastigores L. braziliensis (B). In orange are represented ncRNA genes, while, in purple, the co-expressed protein-coding genes. C. Top 10 biological processes terms assigned to the ncRNAs in each developmental stage based on the co-expressed protein-coding genes.