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An axonemal intron splicing program sustains Plasmodium male development

ABSTRACT: To gain further insights into the post-transcriptional regulation process in the male sexual stage of Plasmodium, we focused on RBPm1, the most specifically expressed RNA-binding protein in the male gametocyte of P. yoelii, for in-depth investigation. In the first part of this study, we identified RBPm1 as a splicing activator, facilitating the splicing of 30 introns among 26 genes in the male gametocytes. Next, we sought to elucidate how RBPm1 exerts its splicing-promoting function. To achieve this, we performed biotin-based proximity labeling experiments on RBPm1, identifying 113 proteins that interact with RBPm1. Among these proteins, all 13 subunits of the spliceosome E complex were present, while other components of the splicing cycle were not significantly enriched. This suggests that RBPm1 participates in the initiation step of RNA splicing by interacting with the E-complex to promote intron splicing.

ORGANISM(S): Plasmodium Yoelii Yoelii 17xnl

SUBMITTER: Jing Yuan

PROVIDER: PXD044094 | iProX | Wed Jul 26 00:00:00 BST 2023

REPOSITORIES: iProX

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Project description:CLAMP transcription factor is a GA binding protein that recruits the male dosage compensation complex MSL to male X-chromosome, thus upregulating transcription level twice that of female X-chromosomes. Interestingly, CLAMP is essential for survival of both male and female individuals. However, what function it plays at the autosomes, especially in females is not known. It is well establised that GA-rich sequences to which CLAMP bind have evolved from polypyrimidine tracts that regulate splicing. Also, MALDI-MS data shows that CLAMP binds to RNA binding proteins involved in sex-specific splicing like, Squid. Furthermore, as part of MSL complex CLAMP interacts with MLE, a RNA helicase which is a known component of spliceosome complex conserved from flies to humans. Thus, we hypothesized that CLAMP plays a role in sex-spcific splicing. Given that CLAMP is a transcription factor, understanding mechanism of how it regulates splicing will add to our understanding about how regulators of gene expression shape the transcriptome differentially between males and females. We tested our hypothesis by identifying CLAMP dependent female and male sex-specific splicing events by analysing RNA-seq data from control as well as clampnull mutant male and female third instar larvae (L3) using suppa based time2splice pipeline (https://github.com/ashleymaeconard/time2splice). We identified 189 and 211 CLAMP dependent splicing events in female and male L3. Out of these 139 were female sex-specific splicing whereas 161 was male-sex-specific splicing. 50 splicing events identified as CLAMP dependent were non-specific to any particular sex, and were affect in both the sexes. Interestingly, we identified splicing of master regulator of sex-determination sxl affected by absence of CLAMP in females whereas splicing of one of the sex-determination pathway effectors, tra regulated by CLAMP in males. These findings indicate CLAMP plays important role in determining sex-specific transcript diversity in Drosophila.

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An axonemal intron splicing program sustains Plasmodium male development

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