Functional analyses of mitoribosome 54S subunit devoid of mitochondria-specific protein sequences.

In Saccharomyces cerevisiae, mitoribosomes are composed of a 54S large subunit (mtLSU) and a 37S small subunit (mtSSU). The two subunits altogether contain 73 mitoribosome proteins (MRPs) and two ribosomal RNAs (rRNAs). Although mitoribosomes preserve some similarities with their bacterial counterparts, they have significantly diverged by acquiring new proteins, protein extensions, and new RNA segments, adapting the mitoribosome to the synthesis of highly hydrophobic membrane proteins. In this study, we investigated the functional relevance of mitochondria-specific protein extensions at the C-terminus (C) or N-terminus (N) present in 19 proteins of the mtLSU. The studied mitochondria-specific extensions consist of long tails and loops extending from globular domains that mainly interact with mitochondria-specific proteins and 21S rRNA moieties extensions. The expression of variants devoid of extensions in uL4 (C), uL5 (N), uL13 (N), uL13 (C), uL16 (C), bL17 (N), bL17 (C), bL21 (24), uL22 (N), uL23 (N), uL23 (C), uL24 (C), bL27 (C), bL28 (N), bL28 (C), uL29 (N), uL29 (C), uL30 (C), bL31 (C), and bL32 (C) did not rescue the mitochondrial protein synthesis capacities and respiratory growth of the respective null mutants. On the contrary, the truncated form of the mitoribosome exit tunnel protein uL24 (N) yields a partially functional mitoribosome. Also, the removal of mitochondria-specific sequences from uL1 (N), uL3 (N), uL16 (N), bL9 (N), bL19 (C), uL29 (C), and bL31 (N) did not affect the mitoribosome function and respiratory growth. The collection of mutants described here provides new means to study and evaluate defective assembly modules in the mitoribosome biogenesis process.

COXPD9 in an individual from Puerto Rico and literature review.

Defects of mitoribosome assembly with destabilization of mitochondrial ribosomal proteins and subsequent aberrant mitochondrial translation machinery are one of the emerging categories of human mitochondrial disease. Mitochondrial translation deficiency constitutes a growing cause of combined oxidative phosphorylation deficiency and overall causes a set of clinically heterogeneous multi-systemic diseases. We present here the sixth individual with combined oxidative phosphorylation deficiency-9 (COXPD9) secondary to a likely pathogenic homozygous MRPL3 variant c.571A > C; p.(Thr191Pro). MRPL3 encodes a large mitochondrial ribosome subunit protein, impairing the mitochondrial translation and resulting in multisystem disease. Similar to previously reported individuals, this reported female proband presented with psychomotor retardation, sensorineural hearing loss, hypertrophic cardiomyopathy, failure to thrive, and lactic acidosis. Further, she has additional, previously unreported, features including Leigh syndrome, cataracts, hypotonia, scoliosis, myopathy, exercise intolerance, childhood-onset cardiomyopathy, and microcephaly. This subject is the oldest reported individual with COXPD9. This report also summarizes the clinical and molecular data of the previously reported individuals with COXPD9 to describe the full phenotypic spectrum.

Mitochondrial ribosome bL34 mutants present diminished translation of cytochrome c oxidase subunits.

Saccharomyces cerevisiae mitoribosomes are specialized in the translation of a few number of highly hydrophobic membrane proteins, components of the oxidative phosphorylation system. Mitochondrial characteristics, such as the membrane system and its redox state driven mitoribosomes evolution through great diversion from their bacterial and cytosolic counterparts. Therefore, mitoribosome presents a considerable number of mitochondrial-specific proteins, as well as new protein extensions. In this work we characterize temperature sensitive mutants of the subunit bL34 present in the 54S large subunit. Although bL34 has bacterial homologs, in yeast it has a long 65 aminoacids mitochondrial N-terminal addressing sequence, here we demonstrate that it can be replaced by the mitochondrial addressing sequence of Neurospora crassa ATP9 gene. The bL34 temperature sensitive mutants present lowered translation of mitochondrial COX1 and COX3, which resulted in reduced cytochrome c oxidase activity and respiratory growth deficiency. The sedimentation properties of bL34 in sucrose gradients suggest that similarly to its bacterial homolog, bL34 is also a later participant in the process of mitoribosome biogenesis.