The T. gondii mitoribosome - build from tiny rRNA fragments expressed from mitochondrial genome recombination sites.

Apicomplexan mitochondrial genomes are highly reduced comprising only three open readings frames (ORFs) encoding essential subunits of the oxidative phosphorylation machinery along with genes for ribosomal RNAs (rRNA). Intriguingly, the rRNA genes are not organized as continuous long units, as observed in most other organisms, rather, they are scattered as multiple small fragments throughout the mitochondrial genome. The organization of the Toxoplasma gondii mitochondrial genome is even more striking consisting of 23 sequence blocks that undergo non-random recombination with each other. Recent studies in our lab have unveiled the expression of 34 small RNAs in T. gondii mitochondria with majority of them having homology to rRNA fragments previously described in other Apicomplexans. Surprisingly, several of these small RNAs are encoded at genomic recombination sites leading to the intriguing situation that the sequence at block borders is dually used for different RNAs. We found several instances where small RNAs partially share sequence. In some even more remarkable cases sequence is dually used for a small RNAs and a messenger RNA. We wondered whether these peculiar small RNAs could function as ribosomal RNA, being integrated into mitochondrial ribosomes together with canonical rRNA fragments and ribosomal proteins. Thus, we established a density-based centrifugation approach to study translation in T. gondii mitochondria. For several mitochondrial small RNAs we observed migration into deep fractions of sucrose density gradients. Additionally, putative mitoribosomal proteins of the large and small ribosomal subunit as well as the three mitochondrial mRNAs have shown comparable migration patterns providing the first strong evidence for the presence of polysomes in T. gondii mitochondria. Among the small RNAs found in polysomal fractions are several RNAs that are encoded at genome recombination sites. Outstanding examples are RNA3 and RNA19 which partially share sequence with coxI and coxIII mRNA, respectively. Whether the dual sequence usage for rRNA fragments and mRNAs could be relevant for regulatory processes remains unclear and will be addressed in future experiments. Moreover, our density gradient centrifugation protocol offers a powerful tool for comprehensive studies on the composition and assembly of the T. gondii mitoribosome.