Identification and Validation of Toxoplasma gondii Mitoribosomal Large Subunit Components

Abstract

Mitochondrial ribosomes are fundamental to mitochondrial function, and thus survival, of nearly all eukaryotes. Despite their common ancestry, mitoribosomes have evolved divergent features in different eukaryotic lineages. In apicomplexans, the mitochondrial rRNA is extremely fragmented raising questions about its evolution, protein composition and structure. Apicomplexan mitochondrial translation and the mitoribosomes are essential in all parasites and life stages studied, highlighting mitoribosomes as a promising target for drugs. Still, the apicomplexan mitoribosome is understudied, with one of the obstacles being that its composition is unknown. Here, to facilitate the study of apicomplexan mitoribosomes, we identified and validated components of the mitoribosomal large subunit in the model apicomplexan Toxoplasma gondii.

Small RNAs from Mitochondrial Genome Recombination Sites are Incorporated intoT. gondiiMitoribosomes

“…However, a comprehensive catalog of mitoribosomal constituents is missing, and the extent to which rRNA fragments are part of mitoribosomes remains unanswered. We selected three short RNAs to test for their presence in high-molecular weight complexes by sucrose gradient analysis of enriched mitochondria (Lacombe et al 2019; Shikha et al 2022). We picked sRNAs that were well detected in RNA gel blot experiments (Fig.…”

“…Additionally, Tg mS35 and Tg uL24m coprecipitated with mitochondria-encoded rRNA. Knockdown of Tg mS35, Tg bL35m, Tg bL36m and Tg bL28m resulted in specific loss of activity of one respiratory complex, which is partially encoded in the mitochondrial genome (Lacombe et al, 2019; Shikha et al, 2022). Nevertheless, it remains to be determined whether these large complexes are actively involved in translation.…”

“…Two mitochondrially localized RNA binding proteins from the RAP (RNA-binding domain abundant in apicomplexans) family are also essential for the survival of P. falciparum , although their role in RNA metabolism has not yet been determined (Hollin et al, 2022). In addition, the depletion of nuclear encoded mito-ribosomal proteins of T. gondii (Lacombe et al 2019; Shikha et al 2022) and P. falciparum (Ke et al 2018) led to defects in the assembly of ETC complexes and in parasite proliferation, suggesting that mitochondrial translation is important for parasite survival. Resistance to the antimalarial drug atovaquone in P. falciparum and T. gondii has been linked to mutations in the cob ( cytB ) gene of mitochondria (McFadden et al, 2000; Srivastava et al, 1999; Syafruddin et al, 1999), further supporting the idea of active, essential translation in apicomplexan mitochondria.…”

Small RNAs from mitochondrial genome recombination sites are incorporated into T. gondii mitoribosomes

“…Recently, compelling evidence has surfaced suggesting the existence of ribosome-sized particles, including ribosomal proteins. Using blue-native gel electrophoresis, it was demonstrated that tagged ribosomal proteins Tg mS35, Tg bL12m, Tg uL3m, and Tg uL24m migrate as constituents of a macromolecular complex within the size range of a ribosome ( Lacombe et al, 2019 ; Shikha et al, 2022 ). Additionally, Tg mS35 and Tg uL24m coprecipitated with mitochondria-encoded rRNA.…”

“…Two mitochondrially localized RNA binding proteins from the RAP (RNA-binding domain abundant in apicomplexans) family are also essential for the survival of P. falciparum , although their role in RNA metabolism has not yet been determined ( Hollin et al, 2022 ). In addition, the depletion of nuclear-encoded mitoribosomal proteins of T. gondii ( Lacombe et al, 2019 ; Shikha et al, 2022 ) and P. falciparum ( Ke et al, 2018 ) led to defects in the assembly of ETC complexes and in parasite proliferation, suggesting that mitochondrial translation is important for parasite survival. Resistance to the antimalarial drug atovaquone in P. falciparum and T. gondii has been linked to mutations in the cob ( cytB ) gene of mitochondria ( McFadden et al, 2000 ; Srivastava et al, 1999 ; Syafruddin et al, 1999 ), further supporting the idea of active, essential translation in apicomplexan mitochondria.…”