Authors

IA Fitzmaurice-O’Neill¹; Z Rolande De Laurent¹; O Janha¹; LV Carruthers¹; A TobinD Beraldi¹; K Modrzynska¹;  ¹ University of Glasgow, Institute of Infection, Immunity & Inflammation,, UK;  ² University of Glasgow , UK

Discussion

Plasmodium has a complex life cycle with multiple life stages generated from the relatively compact ~20 Mb genome. Interestingly, despite the general tendency towards the reduction of the genome size, many Plasmodium genes contain introns. Thus, it is highly possible that alternative splicing (AS) contributes to the generation of different life stages and expands the protein landscape of the parasites. While multiple transcript isoforms have previously been observed, very few studies investigate this phenomenon systematically. This is partially due to poor performance of default AS analysis software packages when faced with the specificity of the Plasmodium genome.

We have generated a custom algorithm based on intron/exon junction counts. Initial validation was carried out by generating the list of junctions affected by a previously identified splicing inhibitor (TCMDC-135051), independently defining differentially expressed and differentially spliced transcripts and validating the predicted mechanism of drug action. Using the same approach, we were able to map differential splicing between different life stages of P. berghei: asexual, male/female gametocyte and ookinete. We have identified several stage-specific transcript isoforms involved in sexual differentiation, DNA replication or cell signalling.

To validate the biological significance of these events, we have generated parasite lines expressing different GFP-tagged isoforms of a conserved Plasmodium protein involved in sexual differentiation. Md2 contains two exons interspaced with an intron. Md2 RNA-sequencing data shows differential splicing between male and female gametocytes. We have shown that both GFP tagged isoforms can be translated and result in different protein localisation and parasitic phenotypes. RNA-sequencing analysis confirmed that correct splicing of the gene is required for male sex determination and absence of either isoform results in lines able to produce female gametocytes only. In summary, we have confirmed differential splicing is a key feature of Plasmodium biology and plays key role in the life cycle progression.