Uncovering the role of DEAD-box RNA helicase TgmRHel1 in mitochondrial gene expression

Apicomplexan parasites have a unique mitochondrion with a highly reduced genome. The genome consists of fragmented rRNA genes and only three protein-coding genes: cob (cytochrome b), coxI (cytochrome oxidase subunit I), and coxIII (cytochrome oxidase subunit III). These genes are crucial for the survival of the parasite as they encode components of the electron transport chain at the heart of oxidative phosphorylation. In Toxoplasma gondii the mitochondrial genome is particularly intriguing. It is composed of 23 sequence blocks that occur in non-random combinations and display an even higher degree of fragmentation. Previous work in our group has shown that genome blocks and their combinations are actively transcribed, identifying a set of 34 mitochondrial small RNAs. It is assumed that in T. gondii similarly to the other Apicomplexa, mRNAs and rRNA fragments are transcribed polycistronically and processed into monocistronic forms by nuclear-encoded factors. However, the processes of mitochondrial RNA processing and translation, including their regulation, remain poorly understood. We identified a putative DEAD-box RNA helicase which we named TgmRHel1. TgmRHel1 localises to the T. gondii mitochondrion and we demonstrated it is required for parasite growth and that its absence severely impacts mitochondrial respiration. Further analysis of the mutant revealed depletion in ETC complexes III and IV. Given the compact nature of the T. gondii mitochondrial genome and the extensive fragmentation of rRNAs, we hypothesize that the observed defects are due to TgmRHel1 playing a role in small RNA processing and mitoribosome assembly or in the processing of mRNA to form mature coxI, coxIII, or cob transcripts. To investigate this, we performed sucrose density centrifugation analysis and shown that in the absence of TgmRHel1 mitoribosomes cannot properly assemble. We further examined the effects on mitochondrial RNAs by RT-qPCR and small RNA sequencing. Our work will present insights and first characterisation of the RNA helicase involved in mitochondrial gene expression in T. gondii.