Mitoribosome structure with cofactors and modifications reveals mechanism of ligand binding and interactions with L1 stalk.

The mitoribosome translates mitochondrial mRNAs and regulates energy conversion that is a signature of aerobic life forms. We present a 2.2 Å resolution structure of human mitoribosome together with validated mitoribosomal RNA (rRNA) modifications, including aminoacylated CP-tRNAVal. The structure shows how mitoribosomal proteins stabilise binding of mRNA and tRNA helping to align it in the decoding center, whereas the GDP-bound mS29 stabilizes intersubunit communication. Comparison between different states, with respect to tRNA position, allowed us to characterize a non-canonical L1 stalk, and molecular dynamics simulations revealed how it facilitates tRNA transitions in a way that does not require interactions with rRNA. We also report functionally important polyamines that are depleted when cells are subjected to an antibiotic treatment. The structural, biochemical, and computational data illuminate the principal functional components of the translation mechanism in mitochondria and provide a description of the structure and function of the human mitoribosome.

Systematic Analysis of Assembly Intermediates in Yeast to Decipher the Mitoribosome Assembly Pathway.

Studies of yeast mitoribosome assembly have been historically hampered by the difficulty of generating mitoribosome protein-coding gene deletion strains with a stable mitochondrial genome. The identification of mitochondrial DNA-stabilizing approaches allows for the generation of a complete set of yeast deletion strains covering all mitoribosome proteins and known assembly factors. These strains can be used to analyze the integrity and assembly state of mitoribosomes by determining the sedimentation profile of these structures by sucrose gradient centrifugation of mitochondrial extracts, coupled to mass spectrometry analysis of mitoribosome composition. Subsequent hierarchical cluster analysis of mitoribosome subassemblies accumulated in mutant strains reveals details regarding the order of protein association during the mitoribosome biogenetic process. These strains also allow the expression of truncated protein variants to probe the role of mitochondrion-specific protein extensions, the relevance of protein cofactors, or the importance of RNA-protein interactions in functional sites of the mitoribosome. In this chapter, we will detail the methodology involved in these studies.

Mitoribosome Biogenesis.

Mitoribosome biogenesis is a complex and energetically costly process that involves RNA elements encoded in the mitochondrial genome and mitoribosomal proteins most frequently encoded in the nuclear genome. The process is catalyzed by extra-ribosomal proteins, nucleus-encoded assembly factors that act in all stages of the assembly process to coordinate the processing and maturation of ribosomal RNAs with the hierarchical association of ribosomal proteins. Biochemical studies and recent cryo-EM structures of mammalian mitoribosomes have provided hints regarding their assembly. In this general concept chapter, we will briefly describe the current knowledge, mainly regarding the mammalian mitoribosome biogenesis pathway and factors involved, and will emphasize the biological sources and approaches that have been applied to advance the field.

Structure of mitoribosome reveals mechanism of mRNA binding, tRNA interactions with L1 stalk, roles of cofactors and rRNA modifications.

The mitoribosome translates mitochondrial mRNAs and regulates energy conversion that is a signature of aerobic life forms. We present a 2.2 Å resolution structure of human mitoribosome together with validated mitoribosomal RNA (rRNA) modifications, including aminoacylated CP-tRNA Val . The structure shows how mitoribosomal proteins stabilise binding of mRNA and tRNA helping to align it in the decoding center, whereas the GDP-bound mS29 stabilizes intersubunit communication. Comparison between different states, with respect to tRNA position, allowed to characterize a non-canonical L1 stalk, and molecular dynamics simulations revealed how it facilitates tRNA transition in a way that does not require interactions with rRNA. We also report functionally important polyamines that are depleted when cells are subjected to an antibiotic treatment. The structural, biochemical, and computational data illuminate the principal functional components of the translation mechanism in mitochondria and provide the most complete description so far of the structure and function of the human mitoribosome.

The Diseased Mitoribosome.

Mitochondria control life and death in eukaryotic cells. Harboring a unique circular genome, a by-product of an ancient endosymbiotic event, mitochondria maintains a specialized and evolutionary divergent protein synthesis machinery, the mitoribosome. Mitoribosome biogenesis depends on elements encoded in both the mitochondrial genome (the RNA components) and the nuclear genome (all ribosomal proteins and assembly factors). Recent cryo-EM structures of mammalian mitoribosomes have illuminated their composition and provided hints regarding their assembly and elusive mitochondrial translation mechanisms. A growing body of literature involves the mitoribosome in inherited primary mitochondrial disorders. Mutations in genes encoding mitoribosomal RNAs, proteins, and assembly factors impede mitoribosome biogenesis, causing protein synthesis defects that lead to respiratory chain failure and mitochondrial disorders such as encephalo- and cardiomyopathy, deafness, neuropathy, and developmental delays. In this article, we review the current fundamental understanding of mitoribosome assembly and function, and the clinical landscape of mitochondrial disorders driven by mutations in mitoribosome components and assembly factors, to portray how basic and clinical studies combined help us better understand both mitochondrial biology and medicine.

Dipeptide repeat proteins inhibit homology-directed DNA double strand break repair in C9ORF72 ALS/FTD.

BACKGROUND: The C9ORF72 hexanucleotide repeat expansion is the most common known genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two fatal age-related neurodegenerative diseases. The C9ORF72 expansion encodes five dipeptide repeat proteins (DPRs) that are produced through a non-canonical translation mechanism. Among the DPRs, proline-arginine (PR), glycine-arginine (GR), and glycine-alanine (GA) are the most neurotoxic and increase the frequency of DNA double strand breaks (DSBs). While the accumulation of these genotoxic lesions is increasingly recognized as a feature of disease, the mechanism(s) of DPR-mediated DNA damage are ill-defined and the effect of DPRs on the efficiency of each DNA DSB repair pathways has not been previously evaluated. METHODS AND RESULTS: Using DNA DSB repair assays, we evaluated the efficiency of specific repair pathways, and found that PR, GR and GA decrease the efficiency of non-homologous end joining (NHEJ), single strand annealing (SSA), and microhomology-mediated end joining (MMEJ), but not homologous recombination (HR). We found that PR inhibits DNA DSB repair, in part, by binding to the nucleolar protein nucleophosmin (NPM1). Depletion of NPM1 inhibited NHEJ and SSA, suggesting that NPM1 loss-of-function in PR expressing cells leads to impediments of both non-homologous and homology-directed DNA DSB repair pathways. By deleting NPM1 sub-cellular localization signals, we found that PR binds NPM1 regardless of the cellular compartment to which NPM1 was directed. Deletion of the NPM1 acidic loop motif, known to engage other arginine-rich proteins, abrogated PR and NPM1 binding. Using confocal and super-resolution immunofluorescence microscopy, we found that levels of RAD52, a component of the SSA repair machinery, were significantly increased iPSC neurons relative to isogenic controls in which the C9ORF72 expansion had been deleted using CRISPR/Cas9 genome editing. Western analysis of post-mortem brain tissues confirmed that RAD52 immunoreactivity is significantly increased in C9ALS/FTD samples as compared to controls. CONCLUSIONS: Collectively, we characterized the inhibitory effects of DPRs on key DNA DSB repair pathways, identified NPM1 as a facilitator of DNA repair that is inhibited by PR, and revealed deficits in homology-directed DNA DSB repair pathways as a novel feature of C9ORF72-related disease.