Characterisation of human ERAL1 as an essential mitochondrial protein

Mitochondria are cellular organelles that are present in all nucleated eukaryotic cells and contain their own genome (mtDNA). Mitochondrial DNA encodes beside 13 polypeptides and 22 tRNAs, 2 ribosomal RNAs. All mtDNA encoded proteins are synthesized within the mitochondrial matrix and essential parts of intra membrane multi-enzyme complexes, involved in oxidative phosphorylation. Mitochondrial protein synthesis is therefore essential for life. This process, however, is still poorly understood. Around 100 mitoribosomal proteins, initiation and elongations factors are involved in mitochondrial translation but the exact mechanisms of how the mitochondrial 55S monosome and the constituted subunits assemble remain obscure. In an attempt to identify factors that play a role in mitoribosome assembly, proteomic analyses of affinity purified complexes using mtRRF were performed (Rorbach et al., 2008). One identified protein was ERAL1, a KH domain containing GTPase with sequence similarity to Era, a eubacterial protein involved in maturation of the 16SrRNA. SiRNA mediated ERAL1 depletion experiments in human cell lines were established and used to investigate the molecular function of the protein. As data in this thesis show, ERAL1 is a mitochondrial protein and is essential for mammalian cells. ERAL1 acts in mitochondria as a 12S-rRNA chaperone via binding at a conserved stem loop structure close to the 3’ terminus of the 12S-rRNA and loss affects the assembly of the small mitoribosomal subunit. Depletion of ERAL1 causes its major growth phenotype, partly by inducing apoptosis. Thus the mitochondrial oxidative phosphorylation machinery is not affected during ERAL1 depletion. ERAL1 is therefore an essential protein in eukaryotic cells, involved in 12S-rRNA maintenance.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Inhibition of proteasome rescues a pathogenic variant of respiratory chain assembly factor COA7.

Nuclear and mitochondrial genome mutations lead to various mitochondrial diseases, many of which affect the mitochondrial respiratory chain. The proteome of the intermembrane space (IMS) of mitochondria consists of several important assembly factors that participate in the biogenesis of mitochondrial respiratory chain complexes. The present study comprehensively analyzed a recently identified IMS protein cytochrome c oxidase assembly factor 7 (COA7), or RESpiratory chain Assembly 1 (RESA1) factor that is associated with a rare form of mitochondrial leukoencephalopathy and complex IV deficiency. We found that COA7 requires the mitochondrial IMS import and assembly (MIA) pathway for efficient accumulation in the IMS We also found that pathogenic mutant versions of COA7 are imported slower than the wild-type protein, and mislocalized proteins are degraded in the cytosol by the proteasome. Interestingly, proteasome inhibition rescued both the mitochondrial localization of COA7 and complex IV activity in patient-derived fibroblasts. We propose proteasome inhibition as a novel therapeutic approach for a broad range of mitochondrial pathologies associated with the decreased levels of mitochondrial proteins.Narodowe Centrum Nauki (NCN) NCN 2012/05/B/NZ3/00781NCN 2015/19/B/NZ3/03272 Deutsche Forschungsgemeinschaft (DFG) SFB1190 (P13) Fundacja na rzecz Nauki Polskiej (FNP) TEAM TECH CORE FACILITY/2016‐2/2MAB/2017/2COP/01/2016 Ministerstwo Nauki i Szkolnictwa Wyższego (MNiSW) Ideas Plus programme 000263 RCUK|Medical Research Council (MRC) MC_UU_00015/5 EC|FP7|FP7 Ideas: European Research Council (FP7 Ideas) FP7‐322424339580 Institut de France Telethon Italy GTB1200

Human ERAL1 is a mitochondrial RNA chaperone involved in the assembly of the 28S small mitochondrial ribosomal subunit

The bacterial Ras-like protein Era has been reported previously to bind 16S rRNA within the 30S ribosomal subunit and to play a crucial role in ribosome assembly. An orthologue of this essential GTPase ERAL1 (Era G-protein-like 1) exists in higher eukaryotes and although its exact molecular function and cellular localization is unknown, its absence has been linked to apoptosis. In the present study we show that human ERAL1 is a mitochondrial protein important for the formation of the 28S small mitoribosomal subunit. We also show that ERAL1 binds in vivo to the rRNA component of the small subunit [12S mt (mitochondrial)-rRNA]. Bacterial Era associates with a 3′ unstructured nonanucleotide immediately downstream of the terminal stem–loop (helix 45) of 16S rRNA. This site contains an AUCA sequence highly conserved across all domains of life, immediately upstream of the anti-Shine–Dalgarno sequence, which is conserved in bacteria. Strikingly, this entire region is absent from 12S mt-rRNA. We have mapped the ERAL1-binding site to a 33 nucleotide section delineating the 3′ terminal stem–loop region of 12S mt-rRNA. This loop contains two adenine residues that are reported to be dimethylated on mitoribosome maturation. Furthermore, and also in contrast with the bacterial orthologue, loss of ERAL1 leads to rapid decay of nascent 12S mt-rRNA, consistent with a role as a mitochondrial RNA chaperone. Finally, whereas depletion of ERAL1 leads to apoptosis, cell death occurs prior to any appreciable loss of mitochondrial protein synthesis or reduction in the stability of mitochondrial mRNA

Ribosome-Targeting Antibiotics Impair T Cell Effector Function and Ameliorate Autoimmunity by Blocking Mitochondrial Protein Synthesis

While antibiotics are intended to specifically target bacteria, most are known to affect host cell physiology. In addition, some antibiotic classes are reported as immunosuppressive for reasons that remain unclear. Here, we show that Linezolid, a ribosomal-targeting antibiotic (RAbo), effectively blocked the course of a T cell mediated autoimmune disease. Linezolid and other RAbos were strong inhibitors of T helper-17 cell effector function in vitro, showing that this effect was independent of their antibiotic activity. Perturbing mitochondria! translation in differentiating T cells, either with RAbos or through the inhibition of mitochondria! elongation factor G1 (mEF-G1) progressively compromised the integrity of the electron transport chain. Ultimately, this led to deficient oxidative phosphorylation, diminishing nicotinamide adenine dinucleotide concentrations and impairing cytokine production in differentiating T cells. In accordance, mice lacking mEF-G1 in T cells were protected from experimental autoimmune encephalomyelitis, demonstrating that this pathway is crucial in maintaining T cell function and pathogenicity

MITRAC15/COA1 promotes mitochondrial translation in a ND2 ribosome–nascent chain complex

The mitochondrial genome encodes for thirteen core subunits of the oxidative phosphorylation system. These proteins assemble with imported proteins in a modular manner into stoichiometric enzyme complexes. Assembly factors assist in these biogenesis processes by providing co-factors or stabilizing transient assembly stages. However, how expression of the mitochondrial-encoded subunits is regulated to match the availability of nuclear-encoded subunits is still unresolved. Here, we address the function of MITRAC15/COA1, a protein that participates in complex I biogenesis and complex IV biogenesis. Our analyses of a MITRAC15 knockout mutant reveal that MITRAC15 is required for translation of the mitochondrial-encoded complex I subunit ND2. We find that MITRAC15 is a constituent of a ribosome-nascent chain complex during ND2 translation. Chemical crosslinking analyses demonstrate that binding of the ND2-specific assembly factor ACAD9 to the ND2 polypeptide occurs at the C-terminus and thus downstream of MITRAC15. Our analyses demonstrate that expression of the founder subunit ND2 of complex I undergoes regulation. Moreover, a ribosome-nascent chain complex with MITRAC15 is at the heart of this process.peerReviewe

Ribosome-Associated Mba1 Escorts Cox2 from Insertion Machinery to Maturing Assembly Intermediates

The three conserved core subunits of the cytochrome c oxidase are encoded by mitochondria in close to all eukaryotes. The Cox2 subunit spans the inner membrane twice, exposing the N and C termini to the intermembrane space. For this, the N terminus is exported cotranslationally by Oxa1 and subsequently undergoes proteolytic maturation in Saccharomyces cerevisiae. Little is known about the translocation of the C terminus, but Cox18 has been identified to be a critical protein in this process. Here we find that the scaffold protein Cox20, which promotes processing of Cox2, is in complex with the ribosome receptor Mba1 and translating mitochondrial ribosomes in a Cox2-dependent manner. The Mba1-Cox20 complex accumulates when export of the C terminus of Cox2 is blocked by the loss of the Cox18 protein. While Cox20 engages with Cox18, Mba1 is no longer present at this stage. Our analyses indicate that Cox20 associates with nascent Cox2 and Mba1 to promote Cox2 maturation cotranslationally. We suggest that Mba1 stabilizes the Cox20-ribosome complex and supports the handover of Cox2 to the Cox18 tail export machinery.peerReviewe

The mitochondrial TMEM177 associates with COX20 during COX2 biogenesis.

The three mitochondrial-encoded proteins, COX1, COX2, and COX3, form the core of the cytochrome c oxidase. Upon synthesis, COX2 engages with COX20 in the inner mitochondrial membrane, a scaffold protein that recruits metallochaperones for copper delivery to the CuA-Site of COX2. Here we identified the human protein, TMEM177 as a constituent of the COX20 interaction network. Loss or increase in the amount of TMEM177 affects COX20 abundance leading to reduced or increased COX20 levels respectively. TMEM177 associates with newly synthesized COX2 and SCO2 in a COX20-dependent manner. Our data shows that by unbalancing the amount of TMEM177, newly synthesized COX2 accumulates in a COX20-associated state. We conclude that TMEM177 promotes assembly of COX2 at the level of CuA-site formation.peerReviewe

Towards Adaptive Social Comparison for Education

Informing students about their progress in comparison to their peers has been widely used in educational research as a strong motivational factor, effective gamification technique and means for adaptive guidance to learning material. A typical social comparison interface helps students weight their individual levels against the average levels of other students. However, such uniform approach may not be effective for every category of students and every learning situation. Underachieving students might find the displayed social goal impossible, while overachieving students might decide that the learning goal has been attained and stop investing time and efforts. An alternative approach is an adaptive social comparison strategy that chooses different levels of the social goal for different categories of students. This paper presents one of the first steps towards developing such a strategy