Dr Stuart Ralph
The University of Melbourne
Alternative splicing in human parasites
RNAseq experiments made possible by next generation sequencing technologies have revealed that alternative splicing is widespread in multicellular organisms, and contributes to tissue differentiation and cellular identity in humans and other animals. Alternative splicing in single celled protists is less well-characterised and its function is unclear. We have applied a combination of direct, long-read MinION nanopore sequencing of mRNA and high-coverage Illumina sequencing of cDNA to survey alternative splicing in Plasmodium and Toxoplasma parasites - the causative agents of significant human diseases. These RNAseq experiments reveal considerable alternative splicing in full length transcripts, and major differences in splicing between life stages. Although some alternative splicing results in novel protein isoforms, many alternative transcripts have premature stop codons and appear unlikely to result in translated products. The Serine/Arginine-rich (SR) family are the major regulators of alternative splicing, and we have identified multiple SR family in each apicomplexan group studied. We have now modulated parasite SR proteins through knockout, knockdown and overexpression in Plasmodium berghei and Toxoplasma gondii. Disruption of some SR proteins leads to defects in asexual proliferative stages, while disruption of other family members leads to blocks in differentiation between life stages. RNAseq analysis of Toxoplasma and Plasmodium SR mutant parasites reveals major transcriptome-wide changes in alternative splicing. Our data reveal alternative splicing as a highly regulated element of apicomplexan gene expression that is central to proliferation and differentiating. We consider this regulation to be analogous to the changes in alternative splicing required for tissue differentiation and identity in multicellular animals.
Stuart Ralph is an Associate Professor in the Department of Biochemistry and Molecular Biology in the Bio21 Institute at The University of Melbourne. His interests include the molecular and cell biology of apicomplexan parasites, particularly Plasmodium and Toxoplasma, and aspects of this biology that may be targeted by chemotherapeutic interventions. Stuart is interested in understanding the essential housekeeping function of gene expression in these parasites and identifying how they differ from their animal hosts. His laboratory studies parasite gene transcription, post transcriptional regulation and protein translation, and characterises inhibitors of these processes. Dr Ralph joined the University of Melbourne in 2007 after postdoctoral research positions at the Pasteur Institute in Paris and the Walter and Eliza Hall Institute in Melbourne.