Poster Presentation First Malaria World Congress 2018

Investigating alternative splicing in stage differentiation of apicomplexan parasites (#325)

Vern Lee 1 2 , Lee Yeoh 2 , Chris Tonkin 1 3 , Stuart Ralph 2
  1. Infection and Immunity, Walter and Eliza Hall Institute of Medical Research, Melbourne, VIC, Australia
  2. Biochemistry and Molecular Biology, The University of Melbourne, Parkville, VIC, Australia
  3. Medical Biology, University of Melbourne, Melbourne, VIC, Australia

In metazoans, alternative splicing is a well-established phenomenon that regulates multiple layers of gene regulation. Recent studies demonstrate that alternative splicing is more common than previously thought in some apicomplexans, particularly Plasmodium and Toxoplasma. One of the most studied alternative-splicing factor is the serine/arginine-rich (SR) protein family. Phosphorylated SR proteins localise to subnuclear regions called speckles, where splicing factors are assembled and stored. Previous work on P. berghei indicates that alternative splicing regulates gender differentiation into sexual gametocytes, and that ablation of one of the SR proteins produces a gender specific defect in male gametocytes. We are now extending these studies to test whether other SR proteins and SR kinases are required for other steps of stage progression in T. gondii and P. falciparum. Taking advantage of CRISPR/Cas technologies, we have generated constitutive and inducible gene knockouts of these proteins in T. gondii. Importantly, ablation of these proteins appear to produce stage specific defects in the parasites, mirroring the P. berghei study. Current efforts are underway to investigate the transcriptional landscape of these parasite using RNA-seq. Traditionally, transcriptomic studies employing RNA-seq have relied on short read technologies (454, Illumina). Despite the power of very high sequencing depth, the short reads presents a limitation in studying the complexity of alternative splicing in individual transcripts. We are now applying third generation sequencing technologies with longer reads, including Oxford Nanopore and PacBio, which should allow us to obtain a fuller picture of splicing on individual, full length transcripts information than was previously possible. Preliminary results from utilising Oxford Nanopore indicates that such is the case, and we anticipate that these studies will provide a better understanding of how alternative splicing modulates stage specific differentiation in apicomplexan parasites.