Single genes are frequently capable of generating multiple mRNAs, coding for alternative protein isoforms, via a process known as alternative splicing. These transcripts generally arise when the spliceosome binds to alternative 5' or 3' splice sites of introns. This phenomenon is widespread in metazoans, and observed in over 90% of human genes. Recent data suggest it may also be common in Apicomplexa. These parasites have small genomes, and economy of DNA appears to be evolutionarily favoured in this phylum. Thus, alternative splicing might play an even more important role for these organisms than metazoans.
We are investigating the mechanism of alternative splicing in Plasmodium spp. and Toxoplasma gondii, and have localised four homologues of ASF/SF2 (alternative-splicing factor/splicing factor 2) to a subnuclear compartment in T. gondii. In addition, we have conditionally overexpressed a homologue in T. gondii using the ddFKBP-Shld1 system. Induced overexpression is deleterious to growth, and perturbation of some alternatively-spliced genes was detected by qRT-PCR.
We also performed high-throughput RNA-seq on our mutant, to determine what splicing is regulated by this homologue. Current RNA-seq algorithms are poorly suited for apicomplexans, which have more compact genomes than metazoans. Hence, we complemented existing tools by writing new programs that address this deficiency. Alternative splicing was perturbed in over 30% of genes in our mutant, and we are working to determine the biological consequences of perturbation.