Mitochondrial RNA Research Articles

Single cell RNA sequencing of hematopoietic cells in fresh and frozen human atheroma tissue.

Single-cell RNA sequencing (scRNA-seq) is a powerful method for exploring the cellular heterogeneity within human atheroma but typically requires fresh tissue to preserve cell membrane integrity, limiting the feasibility of large-scale biobanking for later analysis. The aim of this study was to determine whether cryopreservation of fragile and necrotic atheroma tissue affects the viability and transcriptomic profiles of hematopoietic cells in subsequent scRNA-seq analysis, enabling the use of cryopreserved atheroma samples for future research. We performed scRNA-seq on five paired fresh and cryopreserved atheroma samples - three from coronary arteries and two from carotid arteries. Each sample was enzymatically digested, sorted for CD45+ hematopoietic cells, and processed using the 10X Genomics scRNA-seq workflow. Half of each sample was processed immediately, while the other half was cryopreserved in liquid nitrogen for an average of five weeks before thawing and processing. In carotid artery samples, we noted the absence of LYVE1+ macrophages, likely due to the loss of the adventitial layer during endarterectomy procedures. Our results indicated that cryopreservation modestly affected cellular integrity, leading to an increase in the relative abundance of mitochondrial RNA in frozen samples. Minimal differences were observed between fresh and cryopreserved samples in uniquely detected transcripts, cell clustering, or transcriptional profiles within hematopoietic populations. Our study demonstrates that cryopreserved human atheroma samples can be successfully profiled using scRNA-seq, with comparable transcriptomic data to that obtained from fresh samples. These findings suggest that cryopreservation is a viable method for biobanking atheroma tissues, facilitating large-scale studies without the need for immediate sample processing.

Diversification outbreaks and dynamics of Asian leaf-litter frogs, genus Leptobrachella (Anura, Megophryidae), with the description of a new species from Guizhou Province, China

The uplift of the Qinghai-Tibet Plateau and the Indochina extrusion are two of the most prominent consequences of the India–Asia collision. These two geologic events greatly altered topography and drainage patterns that, in turn, affected the regional climate, the landscape and the evolution of biodiversity. Despite this, little is known about how orogeny and climate affect the evolution of biodiversity, especially the dynamics of diversification, including origins, peaks and endings. Here, we performed phylogenetic and biogeographic analyses of Leptobrachella distributed in Southeast Asia and southern China, based on mitochondrial 16S ribosomal RNA. The results revealed that Leptobrachella may be roughly divided into Clade I from south of the Indo-Burma and Clade II from central and northern Indo-Burma and southern China. We then investigated the diversification of Leptobrachella over time through biogeographic meta-analyses. We showed that the speciation of Leptobrachella was dominated by in situ diversification that was most likely associated with the uplift of the Qinghai–Tibet Plateau, the Indochina extrusion and the intensification of the Asian monsoon and that diversification may have been less influenced by temperature. In situ diversification experienced three small accelerated phases and one decelerated phase initiated at ~ 32 Ma, with a sharp increase at ~ 15 Ma, a peak at ~ 8.7 Ma and a gradual decline after ~ 6 Ma and the peaks of diversification were asynchronous in Southeast Asia and southern China. Our results suggest a three-phase scenario for the diversification of Leptobrachella, with periods of acceleration and deceleration at every stage, a pattern consistent with the Indochina extrusion, the uplift of the Qinghai–Tibet Plateau and the intensification of the Asian monsoon since the Oligocene. This study highlights how biogeographic meta-analyses can be utilised to estimate diversification history in taxa lacking sufficient molecular markers to quantify the impact of orogeny and climatic shifts on diversification processes. In addition, we also identified four undescribed species and described one new species, Leptobrachella xishuiensissp. nov., from Xishui County, Guizhou Province, China.

RNA 5-methylcytosine marks mitochondrial double-stranded RNAs for degradation and cytosolic release

RNA 5-methylcytosine marks mitochondrial double-stranded RNAs for degradation and cytosolic release

Correcting a pathogenic mitochondrial DNA mutation by base editing in mice.

Primary mitochondrial disorders are most often caused by deleterious mutations in the mitochondrial DNA (mtDNA). Here, we used a mitochondrial DddA-derived cytosine base editor (DdCBE) to introduce a compensatory edit in a mouse model that carries the pathological mutation in the mitochondrial transfer RNA (tRNA) alanine (mt-tRNAAla) gene. Because the original m.5024C→T mutation (G→A in the mt-tRNAAla) destabilizes the mt-tRNAAla aminoacyl stem, we designed a compensatory m.5081G→A edit (C→T in the mt-tRNAAla) that could restore the secondary structure of the tRNAAla aminoacyl stem. For this, the DdCBE gene construct was initially tested in an m.5024C→T mutant cell line. The reduced mt-tRNAAla amounts in these cells were increased after editing up to 78% of the mtDNA. Then, DdCBE was packaged in recombinant adeno-associated virus 9 (AAV9) and intravenously administered by retro-orbital injections into mice. Expression of the transduced DdCBE was observed in the heart and skeletal muscle. Total mt-tRNAAla amounts were restored in heart and muscle by the m.5081G→A edit in a dose-dependent manner. Lactate amounts, which were increased in the heart, were also decreased in treated mice. However, the highest dose tested of AAV9-DdCBE also induced severe adverse effects in vivo because of the extensive mtDNA off-target editing that it generated. These results show that although DdCBE is a promising gene therapy tool for mitochondrial disorders, the doses of the therapeutic constructs must be carefully monitored to avoid deleterious off-target editing.

Spatioselective Imaging of Noncoding RNAs in Mitochondria via an Organelle-Specific DNA Assembly Strategy.

Precise imaging of noncoding RNAs (ncRNAs) in specific organelles allows decoding of their functions at subcellular level but lacks advanced tools. Here we present a DNA-based nanobiotechnology for spatially selective imaging of ncRNA (e.g., microRNA (miRNA)) in mitochondria via an organelle-specific DNA assembly strategy. The target miRNA-initiated assembly of DNA hairpins is inhibited by the block of toehold-mediated strand displacement reaction but can be exclusively activated by a mitochondria-encoded ribosomal RNA (rRNA) for hybridization chain reaction, enabling spatial control over miRNA imaging. We demonstrate that the conditionally controlled DNA assembly technology allows for minimization of nonspecific activation and thus improves the spatial precision of miRNA detection. In addition, the strategy is adaptable to visualizing other ncRNAs such as long noncoding RNAs in mitochondria, highlighting the universality of the approach. Overall, this work provides a useful tool for spatially selective imaging of ncRNAs and investigating the functions of organelle-located RNA.

RMRP variants inhibit the cell cycle checkpoints pathway in cartilage‑hair hypoplasia.

Cartilage‑hair hypoplasia (CHH) is an autosomal recessive form of metaphyseal chondrodysplasia caused by RNA component of mitochondrial RNA processing endoribonuclease (RMRP) gene variants; however, its molecular etiology remains unclear. Whole‑exome sequencing was performed to detect possible pathogenic variants in a patient with a typical short stature and sparse hair. A co‑segregation analysis was also conducted and variants in the family members of the patient were confirmed by Sanger sequencing. A novel compound heterozygous variant in RMRP (NR_003051.4: n.‑21_‑2dup and n.197C>T) was identified in the affected patient. Data from 2 years and 4 months of follow‑up showed a positive effect of growth hormone (GH) therapy on height. Subsequently, two gene expression profiles associated with CHH were obtained from the EMBL‑EBI ENA and ArrayExpress databases. Differentially expressed genes between patients with CHH and healthy controls were selected using R software and were subjected to core analysis using ingenuity pathway analysis (IPA) software. IPA core analysis showed that the 'cell cycle checkpoints' was the most prominent canonical pathway, and the top enriched diseases and functions included various types of cancer, immunological diseases, development disorders and respiratory diseases. The integrative analysis displayed that RMRP can regulate the aberrant expression of downstream targets mainly via the transcription factor TP53, which results in the inhibition of 'cell cycle checkpoints'; eventually, functions associated with the CHH phenotype, such as 'growth failure or short stature' are activated. In conclusion, novel disease‑causing genetic variants of RMRP expand the genetic etiology of CHH, which must be clinically differentiated from achondroplasia. The findings of the present study provide new insights into the mechanisms underlying CHH.

Selection of initiator tRNA and start codon by mammalian mitochondrial initiation factor 3 in leaderless mRNA translation.

The mammalian mitochondrial protein synthesis system produces 13 essential subunits of oxidative phosphorylation (OXPHOS) complexes. Translation initiation in mammalian mitochondria is characterized by the use of leaderless messenger RNAs (mRNAs) and non-AUG start codons, where the proofreading function of IF-3mt still remains elusive. Here, we developed a reconstituted mammalian mitochondrial translation system using in vitro transcribed and native mitochondrial transfer RNAs (tRNAs) to investigate IF-3mt's proofreading function. Similar to bacterial IF-3, IF-3mt permits an initiator tRNA to participate in initiation by discriminating the three G-C pairs in its anticodon stem, and by the cognate interactions of its anticodon with the AUG start codon. As a result, IF-3mt promotes the accurate initiation of leaderless mRNAs. Nevertheless, IF-3mt can also facilitate initiation from the non-AUG(AUA) start codon through its unique N- and C-terminal extensions, in concert with the 5-methylcytidine (m5C) or 5-formylcytidine (f5C) modification at the anticodon wobble position of mt-tRNAMet. This is partly because the IF-3mt-specific N- and C-terminal extensions and the KKGK-motif favor leaderless mRNA initiation and relax non-AUG start codon discrimination. Analyses of IF-3mt-depleted human cells revealed that IF-3mt indeed participates in translating the open reading frames (ORFs) of leaderless mRNAs, as well as the internal ORFsof dicistronic mRNAs.

Mitochondrial mRNA and the small subunit rRNA in budding yeasts undergo 3'-end processing at conserved species-specific elements.

Respiration in eukaryotes depends on mitochondrial protein synthesis, which is performed by organelle-specific ribosomes translating organelle-encoded mRNAs. Although RNA maturation and stability are central events controlling mitochondrial gene expression, many of the molecular details in this pathway remain elusive. These include cis- and trans-regulatory factors that generate and protect the 3' ends. Here, we mapped the 3' ends of mitochondrial mRNAs of yeasts classified into multiple families of the subphylum Saccharomycotina. We found that the processing of mitochondrial 15S rRNA and mRNAs involves species-specific sequence elements, which we term 3'-end RNA processing elements (3'-RPEs). In Saccharomyces cerevisiae, the 3'-RPE has long been recognized as a conserved dodecamer sequence, which recent studies have shown specifically interacts with the nuclear genome-encoded pentatricopeptide repeat protein Rmd9. We also demonstrate that, analogous to Rmd9 in S. cerevisiae, two Rmd9 orthologs from the Debaryomycetaceae family interact with their respective 3'-RPEs found in mRNAs and 15S rRNA. Thus, Rmd9-dependent processing of mitochondrial RNA precursors may be a common mechanism among the families of the Saccharomycotina subphylum. Surprisingly, we observed that 3'-RPEs often occur upstream of stop codons in complex I subunit mRNAs from yeasts of the CUG-Ser1 clade. We examined two of these mature mRNAs and found that their stop codons are indeed removed. Thus, translation of these stop-codon-less transcripts would require a noncanonical termination mechanism. Our findings highlight Rmd9 as a key evolutionarily conserved factor in both mitochondrial mRNA metabolism and mitoribosome biogenesis in a variety of yeasts.

Identification and validation of mitochondria-related LncRNA signatures as a novel prognostic model for glioma.

A predictive model for long-term survival is needed, and mitochondrial dysfunction is a key feature of cancer metabolism, though its link to glioma is not well understood. The aim of this study was to identify the molecular characteristics associated with glioma prognosis and explore its potential function. We analyzed RNA-seq data from The Cancer Genome Atlas and identified differentially expressed mitochondrial long noncoding RNAs (lncRNAs) using R's 'limma' package. A prognostic model was developed using 10 selected lncRNAs and validated with Cox regression and least absolute shrinkage and selection operator algorithm. The model's efficacy was assessed using Kaplan-Meier and receiver operating characteristic curve analyses, and its correlation with immune cell profiles and drug sensitivity was explored. A 10-mitochondria-related LncRNA signature was generated. The median risk score values are used to classify glioma samples into low-risk and high-risk groups. In breast patients, the signature-based risk score demonstrated a more potent ability to predict survival than conventional clinicopathological features. Furthermore, we noted a substantial disparity in the number of immune cells, including B cells, CD8+T cells, and macrophages, between the two groups. In addition, the high-risk group exhibited lower half-maximal inhibitory concentration values for specific chemotherapy medications, including bortezomib, luminespib, rapamycin, and 5-fluorouracil. Our study elucidates the diagnostic and prognostic value of mitochondria-related-lncRNAs in the promotion, suppression, and treatment of glioma.

Non-coding RNAs in urinary bladder cancer microenvironment: Diagnostic, therapeutic, and prognostic perspective.

Urinary bladder cancer (UBC) is the ninth most common cancer worldwide. Despite the reliance of UBC therapy on definite pathological grading and classifications, the clinical response among patients varies widely. The molecular basis of this type of cancer appeals to considerable research; hence, new diagnostic and therapeutic options are introduced. Convenient keywords were searched in Google Scholar, PubMed, the Egyptian Knowledge Bank (EKB), and Web of Science. The recent era of UBC research is concerned with non-coding RNAs (ncRNAs), predominantly, microRNAs (miRNAs) and long non-coding RNA (lncRNAs). In addition, snoRNAs, PIWI-interacting RNAs, mitochondrial RNAs, circular, and Schistosoma haematobium-related ncRNAs appeared to contribute to the pathogenesis of the UBC. This review underscored the recently studied ncRNAs and their importance in the pathogenesis of UBC. Besides, we introduced the prospectives regarding their diagnostic, therapeutic, and prognostic significance in UBC clinical settings. Conclusion. Oncogenic and oncosuppressor ncRNAs' definite balances and interaction within the TME of UBC are key players in the fate of the tumor. Thus, profiling ncRNA in-depth inspects the TME of the UBC for better clinical insights.

Isolation and Analysis of Mitochondrial Small RNAs from Rat Liver Tissue and HepG2 Cells.

The presence of non-coding RNAs, such as microRNAs (miRNAs), in mitochondria has been reported by several studies. The biological roles and functions of these mitochondrial miRNAs ("mitomiRs") have not been sufficiently characterized, but the mitochondrial localization of miRNAs has recently gained significance due to modified mitomiR-populations in certain states of diseases. Here, we describe the isolation and analysis of mitochondrial RNAs from rat liver tissue and HepG2 cells. The principle of the analysis is to prepare mitochondria by differential centrifugation. Cytosolic RNA contamination is eliminated by RNase A treatment followed by percoll gradient-purification and RNA-extraction. Small RNA content is verified by capillary electrophoresis. Mitochondrial miRNAs are detected by Q-PCR following synthesis of cDNA. After Q-PCR based mitomiR-profiling, the Normfinder algorithm is applied to identify suitable reference miRNAs to use as normalizers for mitochondrial input and data analysis. The described procedure depicts a simple way of isolating and quantifying mitomiRs in tissue and cell culture samples.

First record of a cold-seep squat lobster Munidopsis lauensis Baba and de Saint Laurent, 1992 (Anomura: Galatheoidea) from Indian waters

Abstract The present study reports the first zoogeographical record of the squat lobster Munidopsis lauensis Baba and de Saint Laurent, 1992 from a cold-seep site in the Krishna-Godavari Basin (Bay of Bengal), Northern Indian Ocean. Molecular analysis of the partial mitochondrial gene coding for cytochrome C oxidase subunit I (COI mtDNA), and mitochondrial 16S ribosomal RNA (16S rRNA) validated its identity. The present observation extends its geographical distribution to the Northern Indian Ocean. Additionally, molecular barcodes (COI) were generated for Munidopsis scobina Alcock, 1894, Munidopsis wardeni Anderson, 1896, and Shinkaia crosnieri Baba and Williams, 1998 collected from the Indian Exclusive Economic Zone.

Targeting mitochondrial RNAs enhances the efficacy of the DNA-demethylating agents

Hypomethylating agents (HMAs) such as azacytidine and decitabine are FDA-approved chemotherapy drugs for hematologic malignancy. By inhibiting DNA methyltransferases, HMAs reactivate tumor suppressor genes (TSGs) and endogenous double-stranded RNAs (dsRNAs) that limit tumor growth and trigger apoptosis via viral mimicry. Yet, HMAs show limited effects in many solid tumors despite the strong induction of TSGs and dsRNAs. Here we show that targeting mitochondrial RNAs (mtRNAs) can enhance the HMA-mediated cell death in lung adenocarcinoma cells. We find that HMA treatment accompanies increased mtRNA levels and subsequent enhancement of metabolic activity, resulting in higher ATP production. Compromising the mitochondrial function by downregulating mature mtRNA expression increased cell death by HMAs. We further perform a CRISPR screening on mtRNA processing factors and find that mtRNA polymerase (POLRMT) and ElaC Ribonuclease Z 2 (ELAC2) depleted cells show increased sensitivity to HMAs by suppressing decitabine-triggered enhancement of ATP production. Moreover, we show that a small molecular inhibitor of POLRMT compromises the metabolic activity and synergistically enhances the cytotoxicity of HMAs. Our study unveils the insensitivity to HMAs through the elevation of mtRNAs and suggests mtRNA regulatory factors as potential synergistic targets to improve the therapeutic benefit of HMAs.

The Vsr-like protein FASTKD4 regulates the stability and polyadenylation of the MT-ND3 mRNA.

Expression of the compact mitochondrialgenome is regulated by nuclear encoded, mitochondrially localized RNA-binding proteins (RBPs). RBPs regulate the lifecycles of mitochondrial RNAs from transcription to degradation by mediating RNA processing, maturation, stability and translation. The Fas-activated serine/threonine kinase (FASTK) family of RBPs has been shown to regulate and fine-tune discrete aspects of mitochondrial gene expression. Although the roles of specific targets of FASTK proteins have been elucidated, the molecular mechanisms of FASTK proteins in mitochondrial RNA metabolism remain unclear. Therefore, we resolved the structure of FASTKD4 at atomic level that includes the RAP domain and the two FAST motifs, creating a positively charged cavity resembling that of the very short patch repair endonuclease. Our biochemical studies show that FASTKD4 binds the canonical poly(A) tail of MT-ND3 enabling its maturation and translation. The in vitro role of FASTKD4 is consistent with its loss in cells that results in decreased MT-ND3 polyadenylation, which destabilizes thismessenger RNA in mitochondria.

A new quantitative reverse transcription PCR assay to improve the routine diagnosis of paracoccidioidomycosis.

Paracoccidioides are dimorphic fungal pathogens and the etiological agents of paracoccidioidomycosis (PCM). This severe systemic mycosis is restricted to Latin America, where it has been historically endemic. Currently, PCM presents the fewest diagnostic tools available when compared to other endemic mycoses. The main PCM diagnostic methods also have limitations. Molecular methods using different protocols have been proposed, but are restricted to a few regions. An analytical transversal study was conducted to evaluate a new molecular tool using specimens from patients diagnosed with PCM at a reference center for endemic mycoses in Rio de Janeiro, Brazil. After whole nucleic acid (WNA) extraction, RT-qPCR was performed in two independent simplex reactions, targeting the mitochondrial small subunit ribosomal RNA genes of Paracoccidioides brasiliensis and Paracoccidioides lutzii. Additionally, WNAs from all PCM-related Paracoccidioides species and from 114 other fungal strains, as well as from samples obtained from patients diagnosed with other endemic mycoses and tuberculosis, were also tested for specificity. The RT-qPCR targeting P. brasiliensis successfully amplified genetic material from all tested Paracoccidioides species but not P. lutzii, which is why a specific RT-qPCR was designed. The RT-qPCR efficiency was 1.95 (95%) with 100% analytical specificity for both targets. All included PCM clinical samples were positive (100% sensitivity) for P. brasiliensis, and all yielded negative for P. lutzii. Additionally, all samples collected from patients with other diseases were negative, reinforcing the assay's specificity. In conclusion, this study proposes a new accurate tool to cover gaps, contributing to the molecular diagnosis of this neglected disease.

DRBD18 acts as a transcript-specific RNA editing auxiliary factor in Trypanosoma brucei.

Uridine insertion/deletion (U-indel) RNA editing of mitochondrial transcripts is a posttranscriptional modification in kinetoplastid organisms, resulting in the generation of mature mRNAs from cryptic precursors. This RNA editing process involves a multi-protein complex holoenzyme and multiple accessory factors. Recent investigations have highlighted the pivotal involvement of accessory RNA binding proteins (RBPs) in modulating RNA editing in T. brucei, often in a transcript-specific manner. DRBD18 is a multifunctional RBP that reportedly impacts the stability, processing, export and translation of nuclear-encoded mRNAs. However, mass spectrometry studies report DRBD18-RESC interactions, prompting us to investigate its role in mitochondrial U-indel RNA editing. In this study, we demonstrate the specific and RNase-sensitive interaction of DRBD18 with multiple RESC factors. Depletion of DRBD18 through RNA interference in procyclic form T. brucei leads to a significant reduction in the levels of edited A6 and COIII mitochondrial transcripts, while its overexpression causes a notable increase in the abundance of these edited mRNAs. RNA immunoprecipitation/qRT-PCR analysis indicates a direct role for DRBD18 in A6 and COIII mRNA editing. We also examined the impact of arginine methylation of DRBD18 in the editing process, revealing that the hypomethylated form of DRBD18, rather than the arginine-methylated version, is essential for promoting these editing events. In conclusion, our findings demonstrate that DRBD18 directly affects the editing of A6 and COIII mRNAs, with its function being modulated by its arginine methylation status, marking the first report of a mitochondrial function for this protein and identifying it as a newly characterized RNA editing auxiliary factor.

KRBP72 facilitates ATPase-dependent editing progression through a structural roadblock in mitochondrial A6 mRNA.

Uridine insertion/deletion editing of mitochondrial messenger RNAs (mRNAs) in kinetoplastids entails the coordinated action of three complexes. RNA Editing Catalytic Complexes (RECCs) catalyze the enzymatic reactions, while the RNA Editing Substrate Binding Complex (RESC) and RNA Editing Helicase 2 Complex (REH2C) coordinate interactions between RECCs, mRNAs and hundreds of guide RNAs that direct edited sequences. Additionally, numerous auxiliary factors are required for productive editing of specific mRNAs. Here, we elucidate the role of KRBP72, an editing auxiliary factor of the ABC adenosine triphosphatase (ATPase) family that exhibits RNA-binding activity. In procyclic form Trypanosoma brucei, KRBP72 knockdown leads to a pause in editing at the base of a predicted stem loop structure in adenosine triphosphate synthase subunit 6 (A6) mRNA. Enhanced cross-linking and affinity purification revealed KRBP72 binding sites both within and upstream of this stem loop. KRBP72 ATPase activity is essential for its A6 mRNA editing function; however, its RNA-binding activity is dispensable. KRBP72 interacts with most RESC proteins in an RNase-sensitive manner. By contrast, RESC12A associates with KRBP72 in an RNase-insensitive fashion, and RESC12A promotes KRBP72's interaction with RNA. Hence, KRBP72 ATPase activity facilitates progression of editing through a challenging secondary structure, highlighting this protein's crucial role in A6 mRNA editing.

Human mitochondrial RNA polymerase structures reveal transcription start-site and slippage mechanism.

Human mitochondrial RNA polymerase (POLRMT) and protein factors TFAM and TFB2M assemble on mitochondrial DNA promoters to initiate promoter-specific transcription. We present cryo-EM structures of two initiation complexes, IC3 and slipped-IC3, with fully resolved transcription bubbles containing RNA transcripts starting from +1 and -1 positions, respectively. These structures reveal the mechanisms of promoter melting, start site selection, and slippage synthesis. Promoter melting begins at -4 with base-specific interactions of -4 and -3 template guanines with POLRMT and -1 non-template adenine with TFB2M, stabilizing the bubble and facilitating initiation from +1. Slippage occurs when a synthesized 2-mer RNA shifts to -1; the -1 position is not an alternative start-site. The conserved non-template sequence (-1)AAA(+2) is recognized by a non-template stabilizing loop (K153LDPRSGGVIKPP165) and Y209 from TFB2M and W1026 of POLRMT. The initiation complex on cryo-EM grids exist in equilibrium with apo and dimeric POLRMTs, whose relative concentrations may regulate transcription initiation.

Deafness-associated mitochondrial 12S rRNA mutation reshapes mitochondrial and cellular homeostasis.

Human mitochondrial 12S ribosomal RNA (rRNA) 1555A>G mutation has been associated with aminoglycoside-induced and nonsyndromic deafness in many families worldwide. Our previous investigation revealed that the m.1555A>G mutation impaired mitochondrial translation and oxidative phosphorylation (OXPHOS). However, the mechanisms by which mitochondrial dysfunctions induced by m.1555A>G mutation regulate intracellular signaling for mitochondrial and cellular integrity remain poorly understood. Here, we demonstrated that the m.1555A>G mutation downregulated the expression of nucleus-encoded subunits of complexes I and IV but upregulated the expression of assemble factors for OXPHOS complexes, using cybrids derived from one hearing-impaired Chinese subject bearing the m.1555A>G mutation and from one hearing normal control lacking the mutation. These alterations resulted in the aberrant assembly, instability, and reduced activities of respiratory chain enzyme complexes I, IV, and V, rate of oxygen consumption, and diminished ATP production. Furthermore, the mutant cell lines carrying the m.1555A>G mutation exhibited decreased membrane potential and increased the production of reactive oxygen species. The aberrant assembly and biogenesis of OXPHOS impacted mitochondrial quality controls, including the imbalance of mitochondrial dynamics via increasing fission with abnormal mitochondrial morphology and impaired mitophagy. Strikingly, the cells bearing the m.1555A>G mutation revealed the upregulation of both ubiquitin-dependent and independent mitophagy pathways, evidenced by increasing levels of Parkin, Pink, BNIP3 and NIX, respectively. The m.1555A>G mutation-induced deficiencies ameliorate the cell homeostasis via elevating the autophagy process and upregulating apoptotic pathways. Our findings provide new insights into pathophysiology of mitochondrial deafness arising from reshaping mitochondrial and cellular homeostasis due to 12S rRNA 1555A>G mutation.

Structure and function of the human mitochondrial MRS2 channel.

The human mitochondrial RNA splicing 2 protein (MRS2) has been implicated in Mg2+ transport across mitochondrial inner membranes, thus having an important role in Mg2+ homeostasis critical for mitochondrial integrity and function. However, the molecular mechanisms underlying its fundamental channel properties such as ion selectivity and regulation remain unclear. Here we present a structural and functional investigation of MRS2. Cryo-electron microscopy structures in various ionic conditions reveal a pentameric channel architecture and the molecular basis of ion permeation and potential regulation mechanisms. Electrophysiological analyses demonstrate that MRS2 is a Ca2+-regulated, nonselective channel permeable to Mg2+, Ca2+, Na+ and K+, which contrasts with its prokaryotic ortholog, CorA, operating as a Mg2+-gated Mg2+ channel. Moreover, a conserved arginine ring within the pore of MRS2 functions to restrict cation movements, thus preventing the channel from collapsing the proton motive force that drives mitochondrial adenosine triphosphate synthesis. Together, our results provide a molecular framework for further understanding MRS2 in mitochondrial function and disease.