Discussion
Apicomplexa are a phylum of protozoan, obligate parasites. Most are pathogenic, and infect a wide range of hosts causing diseases in both humans and animals. A particularly well studied apicomplexan is
Toxoplasma gondii which causes toxoplasmosis, a prevalent infectious disease that poses a health risk to immunocompromised individuals and unborn or newborn babies.
Critical mitochondrial complexes in
T. gondii and related parasites, are very different in composition and structure from the commonly studied ophistokonts (e.g. yeast and mammalian cells), representing both a prominent site of biological divergence and a promising target for antiparasitic drug development. The mitochondrial ribosome (mitoribosome) is a particularly important complex mediating the translation of the three essential proteins necessary for the electron transport chain. The structure consists of 124 total proteins, including 55 clade-specific proteins and
~
53 rRNA fragments, highlighting both the highest degree of rRNA fragmentation in any mitoribosome studied thus far and the importance of numerous clade specific proteins including a family of proteins known as RNA binding domain abundant in Apicomplexa - or RAP proteins.
There are 23 predicted RAP proteins in
T. gondii with four RAP proteins found in the mitoribosome. RAP family members were shown and/or postulated to play a role in RNA stability and processing in both apicomplexans and humans. However, little is known about their mechanism of action. Our preliminary studies provide support to the role of a RAP protein in ribosomal RNA stability and/or processing in
T. gondii, providing an opportunity for mechanistic studies. My work aims to tackle this through the identification and characterization this mechanism via tools such as proteomics and SPA cryo-EM.
Initial progress involves three complementary avenues of work: 1) Utilizing CRISPR-Cas9 to introduce a Twin-Strep tag to the C-terminus of the RAP protein 2) Utilising CRISPR-Cas9 to introduce TurboID for the purposes of proximity labelling and 3) Optimisation of mitochondrial isolation for future affinity purification/immunoprecipitation directly from mitochondria to enrich for our protein of interest.
This work should allow us to better understand the function of RNA processing complexes on ribosomal RNA in
Toxoplasma gondii.
