Fig. 4: RBPm1 deficiency causes intron retention and protein loss of axonemal genes.

A A schematic showing the transcriptome analysis of RBPm1-null male gametocytes. DFsc7;ΔRbpm1 is a DFsc7-derived RBPm1 mutant line. DFsc7 (parental) and DFsc7;ΔRbpm1 (mutant) male gametocytes were sorted by FACS for RNA-seq. B Global analysis of differential intron retention identified 30 retained introns (blue dots) in the mutant versus the parental line. Retained introns with log₂FC ≥ 1 and p ≤ 0.05 were further verified manually by visualization in IGV. The p-values were calculated by quasi-likelihood F-test and adjusted by FDR. These introns were originated from 26 genes. Detailed information of these genes and introns is provided in Supplementary Fig. 4A. C List of the 26 genes with intron retention from (B), categorized by protein function. D–I. RT-PCR confirmation of intron retention in 6 selected genes kinesin8b, PF16, dhc6, dlc1, dlc2, and PY17X_1109100. Genomic DNA (gDNA) from 17XNL parasite, complementary DNA (cDNA) from male gametocytes of parental and mutant parasites were analyzed. Exons are indicated by boxes and introns by lines. Three independent experiments with similar results. RT-PCR analysis for all 26 genes is presented in Supplementary Fig. 5A. J–O. Protein expression analysis of the 6 genes shown in (D–I) in male gametocytes after loss of RBPm1. Each endogenous gene was tagged with a 6HA at the C-terminus in both 17XNL and ΔRbpm1 parasites (the schematic in the top left panel), generating two tagged lines. Immunoblot of the 6HA-tagged protein in gametocytes with and without RBPm1 (bottom left panel). IFA of the 6HA-tagged protein in male gametocytes with and without RBPm1 (right panel). x/y at bottom-left represents the number of HA-positive male gametocytes/the total number of male gametocytes tested. Three independent experiments with similar results. Scale bars: 5 µm.