Periodic Reporting for period 2 - Orgasome (Protein synthesis in organelles)
Reporting period: 2020-11-01 to 2022-04-30
The research is focused on understanding the molecular mechanisms of how mitochondrial genetic code is translated into proteins. To describe the process at the molecular level, we take up the challenge of its visualization in atomic detail by employing the most recent advances in cryo--electron microscopy (cryo-EM). The life of all advanced organisms depends on mitochondria that use oxygen to generate the chemical energy supply that sustains cells. This essential process is formed by a series of chemical reactions performed by mitochondrial proteins. Synthesis of mitochondrial proteins is catalyzed by mitoribosomes, which therefore regulate the cell energy budget. Mitoribosomes represent a distinct class of ribosomes that is very little known about, apart from the fact that they have substantial differences from all other known homologous systems. We use the cutting edge cryo-EM to decipher mechanistic information on mitoribosomes in various functional states, including assembly, and from different organisms. A special focus is on parasitic organisms, where mitochondrial translational apparatus is significantly divergent and therefore represents a potential therapeutic target. Hence, we expect to reveal basic principles of protein synthesis in mitochondria, and their potential applications.
Human mitoribosome translates 13 respiratory chain protein-coding mRNAs from the mitochondrial genome. It consists of three rRNAs and at least 82 mitoribosomal proteins, 36 of which are specific to mitochondria. The mitochondrial rRNA is reduced compared to its bacterial counterpart (2512 vs 4568 nucleotides in Escherichia coli), protein components are extended, and mt-tRNA-Val has been incorporated as an additional constituent. The mitoribosomal proteins probably have functional roles in mitochondria-specific aspects of translation, however due to the limited resolution and heterogeneity of the characterized complexes, atomic models are currently limited, and many key elements are described as “unassigned density”. The existing models suggest that the regulatory elements such as the L1 stalk must have been remodeled, and a putative GTPase mS29 might provide the human mitoribosome with an intrinsic activity, while undergoing structural rearrangements for regulation of subunit association. However, lack of experimental data leaves open the questions of the L1 stalk and mS29 structure and function. It is also not known if the assembly of the supernumerary proteins is facilitated by any cofactors.
The mitochondrial rRNA is modified, and dedicated post-transcriptionally activated enzymes provide means for regulation of gene expression and mitoribosome assembly. Due to the central role of the human mitoribosome in cellular energy production, defects in rRNA modification result in fatal clinical syndromes from birth. Some of the bacterial counterpart modifying enzymes are missing from mitochondria, for example methyltransferases RlmA, RlmB, RmsE and pseudouridine synthase RluA, while other mitochondria-specific have evolved. Therefore, the set of rRNA modifications is different. To date, the molecular mechanisms compensating for the lack of bacteria-like modifications remain unknown, and mitochondria-specific rRNA modifications have not been experimentally evidenced in full. For the incorporated tRNAVal, the amino-acetylation state remains unknown.
In addition, mitoribosomes are linked to age-related diseases that are associated with decline in mitochondrial function and biogenesis. Aging-associated pathological changes can be inhibited by polyamine consumption, which also has anti-inflammatory effects in vivo in mouse models. Polyamines have been shown to be essential for mammalian cell differentiation and proliferation through acting on translation, and whether mitoribosomes might be related to this effect and associated with the cellular senescence needs to be explored.
Finally, it is recognized that mitochondrial increased toxicity leading to clinical symptoms of deafness, neuropathy, and myopathy is due to the off-target binding of antimicrobials to the mitoribosome. A particular aminoglycoside, streptomycin is coupled with a bilateral decreased visual acuity with central scotomas and an altered mitochondrial structure. During pregnancy, this secondary mitochondrial effect might be developed into a fetal toxicity that is further transferred into the embryo. To minimize toxic off‐target effects, approaches based on in silico modeling employing high resolution single-particle cryo-EM structures can be used. Although the sensitivity of mitoribosomes to antimicrobials has been documented, no structural information is available, and the molecular interactions have not been shown, thus chemical details remain unknown.
In this work, we combined structural studies of mitoribosomal complexes with mass spectrometry based quantitative RNA analysis, biochemistry, and molecular dynamics simulations. The local resolution of 1.9-2.4 Å allowed to detect iron-sulfur (2Fe-2S) clusters, nicotinamide adenine dinucleotide (NAD), and three types of functional polyamines as native components of a functional human mitoribosome. We also visualize post-transcriptional and post-translational modifications and identify mRNA binding elements, including channel gating, consisting of six mitoribosomal proteins, as well as specific ions and water molecules in the decoding center. Focused classification and computational simulations revealed how tRNA movement is accompanied by the mitochondria-specific L1 stalk and associated proteins. A previously unknown nucleotide binding site is found on mS29, and its role is established via biochemical assays and structural studies with an analogue. Finally, a structure of the small subunit (SSU) in complex with streptomycin reveals its remodeling and fine features of the binding. Together, these data provide a reference for the structure and function of the human mitoribosome in health and disease.
The mitochondrial rRNA is modified, and dedicated post-transcriptionally activated enzymes provide means for regulation of gene expression and mitoribosome assembly. Due to the central role of the human mitoribosome in cellular energy production, defects in rRNA modification result in fatal clinical syndromes from birth. Some of the bacterial counterpart modifying enzymes are missing from mitochondria, for example methyltransferases RlmA, RlmB, RmsE and pseudouridine synthase RluA, while other mitochondria-specific have evolved. Therefore, the set of rRNA modifications is different. To date, the molecular mechanisms compensating for the lack of bacteria-like modifications remain unknown, and mitochondria-specific rRNA modifications have not been experimentally evidenced in full. For the incorporated tRNAVal, the amino-acetylation state remains unknown.
In addition, mitoribosomes are linked to age-related diseases that are associated with decline in mitochondrial function and biogenesis. Aging-associated pathological changes can be inhibited by polyamine consumption, which also has anti-inflammatory effects in vivo in mouse models. Polyamines have been shown to be essential for mammalian cell differentiation and proliferation through acting on translation, and whether mitoribosomes might be related to this effect and associated with the cellular senescence needs to be explored.
Finally, it is recognized that mitochondrial increased toxicity leading to clinical symptoms of deafness, neuropathy, and myopathy is due to the off-target binding of antimicrobials to the mitoribosome. A particular aminoglycoside, streptomycin is coupled with a bilateral decreased visual acuity with central scotomas and an altered mitochondrial structure. During pregnancy, this secondary mitochondrial effect might be developed into a fetal toxicity that is further transferred into the embryo. To minimize toxic off‐target effects, approaches based on in silico modeling employing high resolution single-particle cryo-EM structures can be used. Although the sensitivity of mitoribosomes to antimicrobials has been documented, no structural information is available, and the molecular interactions have not been shown, thus chemical details remain unknown.
In this work, we combined structural studies of mitoribosomal complexes with mass spectrometry based quantitative RNA analysis, biochemistry, and molecular dynamics simulations. The local resolution of 1.9-2.4 Å allowed to detect iron-sulfur (2Fe-2S) clusters, nicotinamide adenine dinucleotide (NAD), and three types of functional polyamines as native components of a functional human mitoribosome. We also visualize post-transcriptional and post-translational modifications and identify mRNA binding elements, including channel gating, consisting of six mitoribosomal proteins, as well as specific ions and water molecules in the decoding center. Focused classification and computational simulations revealed how tRNA movement is accompanied by the mitochondria-specific L1 stalk and associated proteins. A previously unknown nucleotide binding site is found on mS29, and its role is established via biochemical assays and structural studies with an analogue. Finally, a structure of the small subunit (SSU) in complex with streptomycin reveals its remodeling and fine features of the binding. Together, these data provide a reference for the structure and function of the human mitoribosome in health and disease.
Our first direct visualizations not only confirmed that the system is highly specialized, but we also discovered many novel structural elements, including new human mt-rRNA, specific translation activators and unexpected assembly factors. This allows now asking more complex questions regarding the assembly, molecular mechanism, cotranslational processes and evolution. Building on the expertise we will address these important challenges and to expand on the emerging fundamental field of translation in organelles by applying the most recent developments in cryo-EM. By doing so, I aim to provide new fundamental signatures constituting essential biosynthesis in organelles that will hopefully contribute to a ground-breaking research.