Tsp-SL2, Tsp-SL10, and Tsp-SL12 RNAs are structurally and functionally distinct from other T. spiralis spliced leader RNAs. (A) Secondary structure predictions for the T. spiralis SL (Tsp-SL) RNAs that possess a conserved third stem–loop motif shared with C. elegans SL2.1 RNA (orange shading). Full length Tsp-SL2 is shown to indicate the three numbered stem–loops (I, II, III), spliced leader sequence (gray), donor splice site (5′ss; green), and Sm-binding site (green). Only the third stem–loops are shown for Tsp-SL10 and SL12, and P. punctatus SL2. (B) Multidimensional scaling (MDS) plot of Jaccard distances among Tsp-SL read sets (classified with 8 bp minimum match) from three replicate RNA-seq libraries (Tsp-SL1 to Tsp-SL15) based on presence/absence of spliced leader reads at each gene in the T. spiralis genome (BRAKER + TRINITY Tsp-SL-corrected gene annotations; see main text). (C) Hierarchical clustering (Ward's method) of gene-based Jaccard distances among Tsp-SL read sets (Tsp-SL is abbreviated to “SL” for simplicity). Note that SL13+ contains Tsp-SL13, Tsp-SL14, and Tsp-SL15, which are not reliably distinguishable with an 8 bp minimum tail match. (D) Graphical overview of gene-specific contributions to multivariate group separation (linear discriminant analysis) between Tsp-SL read sets containing Tsp-SL2, SL10, or SL12 (orange) versus all other SLs (gray). The score of each read set in the linear discriminant function (LD1) is plotted as rug marks along the x-axis and score densities within the two groups are overlaid on the y-axis. Below, the contribution (variable loading) of each gene to group discrimination along LD1 is plotted and colored according to directionality (mathematical sign). Genes are ordered by contribution in ascending fashion.
