Mutant/mutation
The mutant lacks expression of PF16 (armadillo repeat protein PF16, putative)

Published in: bioRxiv preprint doi: https://doi.org/10.1101/2023.10.07.561333

Protein (function)
All eukaryotic flagella contain an axoneme, a structure consisting of a central apparatus (a pair of microtubules named C1 and C2) encircled by nine doublet microtubules. PF16 is an important Armadillo (ARM)-repeat protein of the central apparatus of eukaryotic flagella. ARM-repeats consist of a ~42-amino acid structurally conserved repeating motif named after the Drosophila segment polarity gene Armadillo (mammalian homologue, b-catenin). Proteins containing ARM-repeats have diverse roles in eukaryotes, including cell signalling, cytoskeletal organisation and regulation of gene expression. Inactivation of the Chlamydomonas PF16 gene led to loss of the central pair of microtubules and abnormal flagellar function, showing that PF16 is required for flagellar motility and stability of the central apparatus C1 microtubule. Disruption of SPAG6, the mouse PF16 orthologue, results in male infertility, due to loss of sperm motility.RNAi of PF16 in the flagellated protozoan Trypanosoma brucei demonstrates a conserved role in flagellum-dependent motility.

Phenotype
Normal production of male and female gametocytes. However, production of male gametes severely impaired (exflaggelation). No oocyst formation.

Additional information
Analysis of a mutant expressing a C-terminal 6xHA-tagged version of RBPm1 (RMgm-5409) or a C-terminal GFP-tagged version of Rbpm1 (RMgm-5410) showed the following:
- Immunofluorescent assay (IFA) of RBPm1::6xHA showed that RBPm1 was expressed only in gametocytes, but not in asexual blood stages, ookinetes, oocysts, and sporozoites. Gametocyte-specific expression of RBPm1 was also observed in Rbpm1::gfp in which the RBPm1 was tagged with GFP.
- To dissect whether RBPm1 is male-specific, the Rbpm1::6HA gametocytes were co-stained with antibodies against α-Tubulin (a male gametocyte marker) and HA. RBPm1 was only detectable in the male gametocytes.
Additionally, RBPm1 was tagged with 6HA (RMgm-5411) in the reporter line DFsc7 (RMgm-4477) which expresses mCherry in females and GFP in males and observed the male specific expression of RBPm1.
- Localization of Rbpm1 in males was nuclear and consistent protein abundance and nuclear localization of RBPm1 during gametogenesis was observed.

Analysis of mutants with the RNA recognition motifs RRM1 (119-190 aa) and RRM2 (203-274 aa) deleted of endogenous RBPm1 in the 17XNL (∆rrm1, RMgm-5412 and ∆rrm2, RMgm-5413) showed the same phenotype as ∆Rbpm1 indicating an essential role of both two RNA recognition motifs in RBPm1 function.

Genetic crosses were performed between ∆Rbpm1 mutant and the male-deficient line Δmap2 or the female-deficient line Δnek4. As expected, the cross between Δmap2 and Δnek4 produced the ookinetes in vitro. The ookinete formation was restored in the ΔRbpm1 parasites that were crossed with Δnek4 but not Δmap2, further confirming the ∆Rbpm1 defects in male gamete formation. 

Aberrant axonemes were formed in activated male gametocytes ΔRbpm1. All the axonemes in ΔRbpm1 showed severe defects with loss of either central singlet microtubules (MTs) or peripheral doublet MTs. Normal DNA synthesis in activated ΔRbpm1male gametocytes.

Evidence is presented that:
- RBPm1 deficiency causes defective intron splicing of axonemal genes (intron retention, IR, genes).
The whole part of intron was retained in the transcripts for most IR genes, while only a N-terminal part of intron was retained for three IR genes.
- All IR genes are male-specific and 8 IR genes are axoneme-related. Twelve out of the 26 genes were selected for further analysis, including 6 annotated (Kinesin8b, PF16, dhc6, dhc7, dlc1, dlc2) and 6 unannotated (PY17X_1109100, PY17X_0521800, PY17X_1311800, PY17X_1323900, PY17X_1357300, PY17X_1335600). Each gene was endogenously tagged at the N- or C-terminus with a 6HA in the 17XNL, generating the HA-tagged lines (Kinesin8b RMgm-5414, PF16 RMgm-5415, dhc6 RMgm-5416, dhc7 RMgm-5417, dlc1 RMgm-5418, dcl2 RMgm-5419, PY17X_1109100 RMgm-5420, PY17X_0521800 RMgm-5421, PY17X_1311800 RMgm-5422, PY17X_1323900 RMgm-5423, PY17X_1357300 RMgm-5424, PY17X_1335600 RMgm-5425). All 12 proteins  were specifically expressed in male gametocytes during parasite life cycle,  in agreement with their transcript profiling. In inactivated gametocytes, these proteins were distributed at cytoplasm, while after activation 11 of 12 proteins displayed axoneme  localization in the flagellating male gametes. These results suggested that RBPm1 controls intron splicing for a group of the axonemal genes.
- To analyze the effect of IR on the axonemal proteins after RBPm1 loss, the Rbpm1 gene was deleted in each of the following tagged lines: kinesin8B::6HA, PF16::6HA, dhc6::6HA, dlc1::6HA, dlc2::6HA, and 1109100::6HA obtaining 6 RBPm1-null lines. In the absence of RBPm1, all 6 6HA-tagged proteins were not detected or under detectable level in male gametocytes compared to the parental counterparts in both IFA and immunoblot. These results demonstrated that RBPm1 deficiency causes protein expression loss of target axonemal genes.
- To confirm the essential roles of P. yoelii Kinesin8B and PF16 in axoneme assembly (as reported in P. berghei), kinesin8b and PF16 genes were disrupted in the 17XNL, obtaining mutant lines Δkinesin8b (RMgm-5426) and ΔPF16 (RMgm-5427). Depletion of kinesin8b or PF16 blocked or severely impaired male gamete formation respectively. Both mutants produced no midgut oocyst in the infected mosquitoes. Ultrastructure analysis of activated male gametocytes revealed that the Δkinesin8b mutant failed to develop “9+2” axoneme with loss of both central and peripheral MTs, while most of the axonemes lost central MTs (shown as “9+0” or “9+1”) in the ΔPF16. Therefore, depletion of Kinesin8B or PF16 phenocopies RBPm1 deficiency in axoneme assembly.
- Intron retention leads to loss of axonemal protein in RBPm1-null male gametocytes
- Intron deletion restores axonemal proteins and partially rectifies axoneme assembly defects in RBPm1-null gametocytes
- RBPm1 interacts with spliceosome E complex and introns of axonemal genes
The endogenous RBPm1 was tagged with a HA::TurboID motif in the 17XNL, generating the line Rbpm1::TurboID (RMgm-5428) to identify RBPm1-interacting proteins in the gametocytes.
- RBPm1 directs splicing of axonemal introns inserted in a reporter gene
- RBPm1 directs splicing of axonemal introns inserted in an endogenous gene
 
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