Fig. 6: RBPm1 interacts with spliceosome E complex and introns of axonemal genes.

A A schematic showing two modified parasite lines generated for searching RBPm1-interacting proteins by TurboID-based proximity labeling and mass spectrometry. The motif of HA::TurboID and T2A::3NLS::HA::TurboID, respectively, was inserted at the C-terminus of the endogenous RBPm1, generating the line Rbpm1::TurboID and the control line Rbpm1::T2A::TurboID. B Volcano plot displaying 113 significantly enriched proteins (pink dot, cutoffs log₂FC ≥ 1 and p ≤ 0.05) in the Rbpm1::TurboID versus Rbpm1::T2A::TurboID. Among them, 13 subunits (red dot) of the early spliceosome E complex were included. The p-values were calculated by two-sided t-test and adjusted by FDR. C Protein interaction analysis between RBPm1 and six spliceosome E complex subunit proteins (U1-70K, U1-A, U1-C, SF1, U2AF1, and U2AF2) from (B). D IFA of 6HA-tagged RBPm1 and 4Myc-tagged U1 snRNP proteins (U1-70K, U1-A, and U1-C) in male gametocytes of three double-tagged parasites. Three independent experiments with similar results. Scale bars: 5 µm. E Co-immunoprecipitation of RBPm1 and U1-70K in gametocytes of the double-tagged parasite U1-70K::4Myc;Rbpm1::6HA. Anti-Myc nanobody was used. Bip as a loading control. Three independent experiments with similar results. F Co-immunoprecipitation of RBPm1 and U1-A in gametocytes of the double-tagged parasite U1-A::4Myc;Rbpm1::6HA. Three independent experiments with similar results. G Co-immunoprecipitation of RBPm1 and U1-C in gametocytes of the double-tagged parasite U1-C::4Myc;Rbpm1::6HA. Three independent experiments with similar results. H Proposed model showing the interaction between RBPm1 and early spliceosome E complex for intron splicing of axonemal genes. I–N. UV-RIP detection of RBPm1 interaction with the retained introns of 6 axonemal genes (kinesin8b, PF16, dhc6, dlc1, dlc2, and PY17X_1109100). A top schematic shows the exon-intron structure of the RBPm1 target axonemal genes. The retained introns are indicated with orange lines, and the genomic regions for qPCR amplicon are shown. UV-RIP was performed in Rbpm1::6HA lines using anti-HA nanobody. Anti-GFP nanobody was used as a control. Bound RNA was analyzed by RT-qPCR. Means ± SEM from three independent experiments, two-sided t-test. O RNA pull-down assay detecting RBPm1 interaction with kinesin8b intron1. A top schematic shows the exon-intron structure of the kinesin8b gene. The retained introns are indicated in orange lines. A biotinylated 500 nt RNA probe I1 (comprising intron1 and its flanking sequences) and a control probe I4 (comprising intron4 and its flanking sequences) were used. Proteins via RNA pull-down were immunoblot with anti-HA antibody. The numbers are the relative intensities of bands in the blot. Histone H3 and Bip were used as negative controls. Two independent experiments with similar results. P RNA pull-down assay detecting RBPm1 interaction with PF16 intron1. A top schematic shows the exon-intron structure of the PF16 gene. The retained introns are indicated in orange lines. A biotinylated 500 nt RNA probe I1 (comprising intron1 and its flanking sequences) and a control probe E1 in exon1 were used. Two independent experiments with similar results.