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Background: SmithRNAs (Small MITochondrial Highly-transcribed RNAs) are a novel class of small RNA molecules that are encoded in the mitochondrial genome and regulate the expression of nuclear transcripts. Initial evidence for their existence came from the Manila clam Ruditapes philippinarum, where they have been described and whose activity has been biologically validated through RNA injection experiments. Current evidence on the existence of these RNAs in other species is based only on small RNA sequencing. As a preliminary step to characterize smithRNAs across different metazoan lineages, a dedicated, unified, analytical workflow is needed. Results: We propose a novel workflow specifically designed for smithRNAs. Sequence data (from small RNA sequencing) uniquely mapping to the mitochondrial genome are clustered into putative smithRNAs and prefiltered based on their abundance, presence in replicate libraries and 5′ and 3′ transcription boundary conservation. The surviving sequences are subsequently compared to the untranslated regions of nuclear transcripts based on seed pairing, overall match and thermodynamic stability to identify possible targets. Ample collateral information and graphics are produced to help characterize these molecules in the species of choice and guide the operator through the analysis. The workflow was tested on the original Manila clam data. Under basic settings, the results of the original study are largely replicated. The effect of additional parameter customization (clustering threshold, stringency, minimum number of replicates, seed matching) was further evaluated. Conclusions: The study of smithRNAs is still in its infancy and no dedicated analytical workflow is currently available. At its core, the SmithHunter workflow builds over the bioinformatic procedure originally applied to identify candidate smithRNAs in the Manila clam. In fact, this is currently the only evidence for smithRNAs that has been biologically validated and, therefore, the elective starting point for characterizing smithRNAs in other species. The original analysis was readapted using current software implementations and some minor issues were solved. Moreover, the workflow was improved by allowing the customization of different analytical parameters, mostly focusing on stringency and the possibility of accounting for a minimal level of genetic differentiation among samples. [ABSTRACT FROM AUTHOR]

Mitochondrial oxidative phosphorylation (OXPHOS) fuels cellular ATP demands. OXPHOS defects lead to severe human disorders with unexplained tissue specific pathologies. Mitochondrial gene expression is essential for OXPHOS biogenesis since core subunits of the complexes are mitochondrial-encoded. COX14 is required for translation of COX1, the central mitochondrial-encoded subunit of complex IV. Here we describe a COX14 mutant mouse corresponding to a patient with complex IV deficiency. COX14M19I mice display broad tissue-specific pathologies. A hallmark phenotype is severe liver inflammation linked to release of mitochondrial RNA into the cytosol sensed by RIG-1 pathway. We find that mitochondrial RNA release is triggered by increased reactive oxygen species production in the deficiency of complex IV. Additionally, we describe a COA3Y72C mouse, affected in an assembly factor that cooperates with COX14 in early COX1 biogenesis, which displays a similar yet milder inflammatory phenotype. Our study provides insight into a link between defective mitochondrial gene expression and tissue-specific inflammation. Dysfunctions of the mitochondrial OXPHOS system lead to severe human disorders. Here, the authors analyzed a mouse mutant that mimics a mitochondrial disorder patient and find that reactive oxygen species trigger RNA release and inflammatory pathway in liver. [ABSTRACT FROM AUTHOR]

The development of safe and effective broad-spectrum antivirals that target the replication machinery of respiratory viruses is of high priority in pandemic preparedness programs. Here, we studied the mechanism of action of a newly discovered nucleotide analog against diverse RNA-dependent RNA polymerases (RdRps) of prototypic respiratory viruses. GS646939 is the active 50 -triphosphate metabolite of a 4ʹ-cyano modified C-adenosine analog phosphoramidate prodrug GS7682. Enzyme kinetics show that the RdRps of human rhinovirus type 16 (HRV-16) and enterovirus 71 incorporate GS646939 with unprecedented selectivity; GS-646939 is incorporated 20-50-fold more efficiently than its natural ATP counterpart. The RdRp complex of respiratory syncytial virus and human metapneumovirus incorporate GS-646939 and ATP with similar efficiency. In contrast, influenza B RdRp shows a clear preference for ATP and human mitochondrial RNA polymerase does not show significant incorporation of GS646939. Once incorporated into the nascent RNA strand, GS646939 acts as a chain terminator although higher NTP concentrations can partially overcome inhibition for some polymerases. Modeling and biochemical data suggest that the 4ʹmodification inhibits RdRp translocation. Comparative studies with GS-443902, the active triphosphate form of the 10 -cyano modified prodrugs remdesivir and obeldesivir, reveal not only different mechanisms of inhibition, but also differences in the spectrum of inhibition of viral polymerases. In conclusion, 1ʹcyano and 4ʹ-cyano modifications of nucleotide analogs provide complementary strategies to target the polymerase of several families of respiratory RNA viruses. [ABSTRACT FROM AUTHOR]

The subfamily Rasborinae is a species-rich group of freshwater fish related to zebrafish; however, its taxonomy remains unclear. We present the complete mitogenome and corresponding polyadenylated mitotranscriptome of Rasbora rasbora (Hamilton 1822) based on long-read and high-coverage Oxford Nanopore Technology sequencing. The mitogenome size, gene content, and gene organization correspond to the typical vertebrate composition, and the mitogenome generates 10 polyadenylated mRNAs. Two alternative polyadenylation sites of ND5 mRNA were detected, one with a 596 nt 3′untranslated region corresponding to the antisense ND6 gene. Polyadenylation also generates seven of the mRNA UAA stop codons. Complete mitogenome sequences, excluding the control region, were carefully aligned for RNA-coding and protein-coding features using 54 available species of the subfamily Rasborinae. The phylogenetic analyses based on maximum likelihood, Bayesian inference, and neighbor-joining tree building methods confirm the transfer of R. rasbora into the Sumatrana species group. The overall phylogeny of the subfamily Rasborinae supports with high confidence some previously observed changes within this subfamily, as well as contradicts some conclusions set by previous studies. [ABSTRACT FROM AUTHOR]

Thirteen-lined ground squirrels (TLGSs) are obligate hibernators that cycle between torpor (low metabolic rate and body temperature) and interbout euthermia (IBE; typical euthermic body temperature and metabolism) from late autumn to spring. Many physiological changes occur throughout hibernation, including a reduction in liver mitochondrial metabolism during torpor, which is reversed during arousal to interbout euthermia. Nuclear-encoded microRNA (miRNA, small posttranscriptional regulator molecules) differ in abundance throughout TLGS hibernation and have been shown to regulate mitochondrial gene expression in mammalian cell culture (where they are referred to as mitomiRs). This study characterized differences in mitomiR profiles from TLGS liver mitochondria isolated during summer, torpor, and IBE, and predicted their mitochondrial targets. Using small RNA sequencing, differentially abundant mitomiRs were identified between hibernation states, and using quantitative PCR analysis, we quantified the expression of predicted mitochondrial mRNA targets. Most differences in mitomiR abundances were seasonal (i.e., between summer and winter) with only one mitomiR differentially abundant between IBE and torpor. Multiple factor analysis (MFA) revealed three clusters divided by hibernation states, where clustering was predominantly driven by mitomiR abundances. Nine of these differentially abundant mitomiRs had predicted mitochondrial RNA targets, including subunits of electron transfer system complexes I and IV, 12S rRNA, and two tRNAs. Overall, mitomiRs were predicted to suppress the expression of their mitochondrial targets and may have some involvement in regulating protein translation in mitochondria. This study found differences in mitomiR abundances between seasons and hibernation states of TLGS and suggests potential mechanisms for regulating the mitochondrial electron transfer system. NEW & NOTEWORTHY: During the hibernation season, thirteen-lined ground squirrels periodically increase metabolism remarkably between torpor and interbout euthermia (IBE). This process involves rapid reactivation of mitochondrial respiration. We predicted that mitochondrial microRNA (mitomiRs) might be altered during this response. We found that the abundance of 38 liver mitomiRs differs based on hibernation state (summer, IBE, and torpor). Small RNA sequencing identified mitomiR profiles, including some mitomiRs that are predicted to bind to mitochondrial RNAs. [ABSTRACT FROM AUTHOR]

Elevated activity of retrotransposons is increasingly recognized to be implicated in a wide range of neurodegenerative and neurodevelopmental diseases, including Down syndrome (DS), which is the most common chromosomal condition causing intellectual disability globally. Previous research by our group has revealed that treatment with lamivudine, a reverse transcriptase inhibitor, improved neurobehavioral phenotypes and completely rescued hippocampaldependent recognition memory in a DS mouse model, Ts65Dn. We hypothesized that retrotransposition rates would increase in the Ts65Dn mouse model, and lamivudine could block retrotransposons. We analyzed the differentially expressed long interspersed element-1 (LINE-1 or L1) mapping on MMU16 and 17, and showed for the first time that retrotransposition could be associated with Ts65Dn's pathology, as misregulation of L1 was found in brain tissues associated with trisomy. In the cerebral cortex, 6 out of 26 upregulated L1s in trisomic treated mice were located in the telomeric region of MMU16 near Ttc3, Kcnj6, and Dscam genes. In the hippocampus, one upregulated L1 element in trisomic treated mice was located near the Fgd4 gene on MMU16. Moreover, two downregulated L1s rescued after treatment with lamivudine were located in the intronic region of Nrxn1 (MMU17) and Snhg14 (MMU7), implicated in a variety of neurodegenerative disorders. To gain further insight into the mechanism of this improvement, we here analyzed the gene expression profile in the hippocampus and cerebral cortex of trisomic mice treated and no-treated with lamivudine compared to their wild-type littermates. We found that treatment with lamivudine rescued the expression of 24% of trisomic genes in the cortex (located on mouse chromosome (MMU) 16 and 17) and 15% in the hippocampus (located in the human chromosome 21 orthologous regions), with important DS candidate genes such as App and Ets2, rescued in both regions. [ABSTRACT FROM AUTHOR]

Aim: Hyperglycemia damages mitochondria and downregulates transcription of mtDNA-encoded genes and the long noncoding RNA LncCytB, causing mitochondrial genomic instability. The genes encoded by mtDNA are transcribed as large polycistronic transcripts, and the 5′ ends of precursor tRNAs are processed by mitochondrial-targeted ribonuclease P (MRPPs). Our aim was to investigate the role of MRPP1 in the downregulation of LncCytB in diabetic retinopathy. Methods: Using human retinal endothelial cells incubated in 20 mM D-glucose for 96 h, the gene expression and mitochondrial localization (immunofluorescence) of MRPP1 and the interaction between MRPP1 and LncCytB (determined by RNA-FISH and RNA immunoprecipitation) were quantified. The results were confirmed in retinal microvessels from streptozotocin-induced diabetic mice and from human donors with documented diabetic retinopathy. Results: Compared to normal glucose, high glucose decreased mRNA and mitochondrial localization of MRPP1 and its interaction with LncCytB. While MRPP1 overexpression prevented glucose-induced decrease in MRPP1–LncCytB interaction, LncCytB expression and mitochondrial damage (reduction in protective nucleoids in mtDNA), MRPP1-siRNA further worsened them. Similar results were obtained from retinal microvessels from diabetic mice and from human donors with diabetic retinopathy. Conclusions: Downregulation of MRPP1 in diabetes suppresses LncCytB transcription, resulting in mitochondrial functional and genomic instability, ultimately leading to the development of diabetic retinopathy. Thus, preventing MRPP1 downregulation has the potential to inhibit retinopathy and prevent the fear of vision loss in diabetic patients. [ABSTRACT FROM AUTHOR]

PARK7, also known as DJ-1, plays a critical role in protecting cells by functioning as a sensitive oxidation sensor and modulator of antioxidants. DJ-1 acts to maintain mitochondrial function and regulate transcription in response to different stressors. In this study, we showed that cell lines vary based on their antioxidation potential under basal conditions. The transcriptome of HEK293 cells was tested following knockdown (KD) of DJ-1 using siRNAs, which reduced the DJ-1 transcripts to only 12% of the original level. We compared the expression levels of 14k protein-coding transcripts and 4.2k non-coding RNAs relative to cells treated with non-specific siRNAs. Among the coding genes, approximately 200 upregulated differentially expressed genes (DEGs) signified a coordinated antiviral innate immune response. Most genes were associated with the regulation of type 1 interferons (IFN) and the induction of inflammatory cytokines. About a quarter of these genes were also induced in cells treated with non-specific siRNAs that were used as a negative control. Beyond the antiviral-like response, 114 genes were specific to the KD of DJ-1 with enrichment in RNA metabolism and mitochondrial functions. A smaller set of downregulated genes (58 genes) was associated with dysregulation in membrane structure, cell viability, and mitophagy. We propose that the KD DJ-1 perturbation diminishes the protective potency against oxidative stress. Thus, it renders the cells labile and responsive to the dsRNA signal by activating a large number of genes, many of which drive apoptosis, cell death, and inflammatory signatures. The KD of DJ-1 highlights its potency in regulating genes associated with antiviral responses, RNA metabolism, and mitochondrial functions, apparently through alteration in STAT activity and downstream signaling. Given that DJ-1 also acts as an oncogene in metastatic cancers, targeting DJ-1 could be a promising therapeutic strategy where manipulation of the DJ-1 level may reduce cancer cell viability and enhance the efficacy of cancer treatments. [ABSTRACT FROM AUTHOR]

Non-coding RNAs (ncRNAs) are a heterogeneous group, in terms of structure and sequence length, consisting of RNA molecules that do not code for proteins. These ncRNAs have a central role in the regulation of gene expression and are virtually involved in every process analyzed, ensuring cellular homeostasis. Although, over the years, much research has focused on the characterization of non-coding transcripts of nuclear origin, improved bioinformatic tools and next-generation sequencing (NGS) platforms have allowed the identification of hundreds of ncRNAs transcribed from the mitochondrial genome (mt-ncRNA), including long non-coding RNA (lncRNA), circular RNA (circRNA), and microRNA (miR). Mt-ncRNAs have been described in diverse cellular processes such as mitochondrial proteome homeostasis and retrograde signaling; however, the function of the majority of mt-ncRNAs remains unknown. This review focuses on a subgroup of human mt-ncRNAs whose dysfunction is associated with both failures in cell cycle regulation, leading to defects in cell growth, cell proliferation, and apoptosis, and the development of tumor hallmarks, such as cell migration and metastasis formation, thus contributing to carcinogenesis and tumor development. Here we provide an overview of the mt-ncRNAs/cancer relationship that could help the future development of new biomedical applications in the field of oncology. [ABSTRACT FROM AUTHOR]

Background: Cell‐free long RNAs in human plasma and extracellular vesicles (EVs) have shown promise as biomarkers in liquid biopsy, despite their fragmented nature. Methods: To investigate these fragmented cell‐free RNAs (cfRNAs), we developed a cost‐effective cfRNA sequencing method called DETECTOR‐seq (depletion‐assisted multiplexed cell‐free total RNA sequencing). DETECTOR‐seq utilised a meticulously tailored set of customised guide RNAs to remove large amounts of unwanted RNAs (i.e., fragmented ribosomal and mitochondrial RNAs) in human plasma. Early barcoding strategy was implemented to reduce costs and minimise plasma requirements. Results: Using DETECTOR‐seq, we conducted a comprehensive analysis of cell‐free transcriptomes in both whole human plasma and EVs. Our analysis revealed discernible distributions of RNA types in plasma and EVs. Plasma exhibited pronounced enrichment in structured circular RNAs, tRNAs, Y RNAs and viral RNAs, while EVs showed enrichment in messenger RNAs (mRNAs) and signal recognition particle RNAs (srpRNAs). Functional pathway analysis highlighted RNA splicing‐related ribonucleoproteins (RNPs) and antimicrobial humoral response genes in plasma, while EVs demonstrated enrichment in transcriptional activity, cell migration and antigen receptor‐mediated immune signals. Our study indicates the comparable potential of cfRNAs from whole plasma and EVs in distinguishing cancer patients (i.e., colorectal and lung cancer) from healthy donors. And microbial cfRNAs in plasma showed potential in classifying specific cancer types. Conclusions: Our comprehensive analysis of total and EV cfRNAs in paired plasma samples provides valuable insights for determining the need for EV purification in cfRNA‐based studies. We envision the cost effectiveness and efficiency of DETECTOR‐seq will empower transcriptome‐wide investigations in the fields of cfRNAs and liquid biopsy. Keypoints: DETECTOR‐seq (depletion‐assisted multiplexed cell‐free total RNA sequencing) enabled efficient and specific depletion of sequences derived from fragmented ribosomal and mitochondrial RNAs in plasma.Distinct human and microbial cell‐free RNA (cfRNA) signatures in whole Plasma versus extracellular vesicles (EVs) were revealed.Both Plasma and EV cfRNAs were capable of distinguishing cancer patients from normal individuals, while microbial RNAs in Plasma cfRNAs enabled better classification of cancer types than EV cfRNAs. [ABSTRACT FROM AUTHOR]

Mitochondria are central to endothelial cell activation and angiogenesis, with the RNA polymerase mitochondrial (POLRMT) serving as a key protein in regulating mitochondrial transcription and oxidative phosphorylation. In our study, we examined the impact of POLRMT on angiogenesis and found that its silencing or knockout (KO) in human umbilical vein endothelial cells (HUVECs) and other endothelial cells resulted in robust anti-angiogenic effects, impeding cell proliferation, migration, and capillary tube formation. Depletion of POLRMT led to impaired mitochondrial function, characterized by mitochondrial depolarization, oxidative stress, lipid oxidation, DNA damage, and reduced ATP production, along with significant apoptosis activation. Conversely, overexpressing POLRMT promoted angiogenic activity in the endothelial cells. In vivo experiments demonstrated that endothelial knockdown of POLRMT, by intravitreous injection of endothelial specific POLRMT shRNA adeno-associated virus, inhibited retinal angiogenesis. In addition, inhibiting POLRMT with a first-in-class inhibitor IMT1 exerted significant anti-angiogenic impact in vitro and in vivo. Significantly elevated expression of POLRMT was observed in the retinal tissues of streptozotocin-induced diabetic retinopathy (DR) mice. POLRMT endothelial knockdown inhibited pathological retinal angiogenesis and mitigated retinal ganglion cell (RGC) degeneration in DR mice. At last, POLRMT expression exhibited a substantial increase in the retinal proliferative membrane tissues of human DR patients. These findings collectively establish the indispensable role of POLRMT in angiogenesis, both in vitro and in vivo. [ABSTRACT FROM AUTHOR]

Mitochondria are cellular powerhouses that generate energy through the electron transport chain (ETC). The mitochondrial genome (mtDNA) encodes essential ETC proteins in a compartmentalized manner, however, the mechanism underlying metabolic regulation of mtDNA function remains unknown. Here, we report that expression of tricarboxylic acid cycle enzyme succinate-CoA ligase SUCLG1 strongly correlates with ETC genes across various TCGA cancer transcriptomes. Mechanistically, SUCLG1 restricts succinyl-CoA levels to suppress the succinylation of mitochondrial RNA polymerase (POLRMT). Lysine 622 succinylation disrupts the interaction of POLRMT with mtDNA and mitochondrial transcription factors. SUCLG1-mediated POLRMT hyposuccinylation maintains mtDNA transcription, mitochondrial biogenesis, and leukemia cell proliferation. Specifically, leukemia-promoting FMS-like tyrosine kinase 3 (FLT3) mutations modulate nuclear transcription and upregulate SUCLG1 expression to reduce succinyl-CoA and POLRMT succinylation, resulting in enhanced mitobiogenesis. In line, genetic depletion of POLRMT or SUCLG1 significantly delays disease progression in mouse and humanized leukemia models. Importantly, succinyl-CoA level and POLRMT succinylation are downregulated in FLT3- mutated clinical leukemia samples, linking enhanced mitobiogenesis to cancer progression. Together, SUCLG1 connects succinyl-CoA with POLRMT succinylation to modulate mitochondrial function and cancer development. Synopsis: Whether mitochondrial DNA function is affected by cellular metabolism remains poorly understood. Here, the Krebs cycle enzyme succinate-CoA ligase (SUCLG1) is shown to restrict succinylation of mitochondrial RNA polymerase (POLRMT) and thereby sustain mitochondrial transcription. This mechanism is relevant for mutant FLT3-driven leukemogenesis. SUCLG1 reduces succinyl-CoA levels to restrict POLRMT succinylation. K622 succinylation suppresses POLRMT activity. FLT3 mutations regulate SUCLG1 to activate POLRMT. Mutant FLT3 alters nuclear transcription to remodel mitochondrial biogenesis. Genetic depletion of POLRMT or SUCLG1 delays leukemia progression in mice and humanized models. Krebs cycle succinate-CoA ligase targets mitochondrial RNA polymerase and mtDNA transcription supporting mutant FLT3-driven blood cancer. [ABSTRACT FROM AUTHOR]

Thyroid cancer diagnosis primarily relies on imaging techniques and cytological analyses. In cases where the diagnosis is uncertain, the quantification of molecular markers has been incorporated after cytological examination. This approach helps physicians to make surgical decisions, estimate cancer aggressiveness, and monitor the response to treatments. Despite the availability of commercial molecular tests, their widespread use has been hindered in our experience due to cost constraints and variability between them. Thus, numerous groups are currently evaluating new molecular markers that ultimately will lead to improved diagnostic certainty, as well as better classification of prognosis and recurrence. In this review, we start reviewing the current preoperative testing methodologies, followed by a comprehensive review of emerging molecular markers. We focus on micro RNAs, long non-coding RNAs, and mitochondrial (mt) signatures, including mtDNA genes and circulating cell-free mtDNA. We envision that a robust set of molecular markers will complement the national and international clinical guides for proper assessment of the disease. [ABSTRACT FROM AUTHOR]

A detailed understanding of how spaceflight affects human health is essential for long-term space exploration. Liquid biopsies allow for minimally-invasive multi-omics assessments that can resolve the molecular heterogeneity of internal tissues. Here, we report initial results from the JAXA Cell-Free Epigenome Study, a liquid biopsy study with six astronauts who resided on the International Space Station (ISS) for more than 120 days. Analysis of plasma cell-free RNA (cfRNA) collected before, during, and after spaceflight confirms previously reported mitochondrial dysregulation in space. Screening with 361 cell surface marker antibodies identifies a mitochondrial DNA-enriched fraction associated with the scavenger receptor CD36. RNA-sequencing of the CD36 fraction reveals tissue-enriched RNA species, suggesting the plasma mitochondrial components originated from various tissues. We compare our plasma cfRNA data to mouse plasma cfRNA data from a previous JAXA mission, which had used on-board artificial gravity, and discover a link between microgravity and the observed mitochondrial responses. It has been reported that a spaceflight causes mitochondrial stress in astronauts. Here the authors suggest that mitochondrial components are released into the plasma during spaceflight as components of CD36-marked extracellular vesicles (EVs). [ABSTRACT FROM AUTHOR]

Incidences of drug-resistant tuberculosis have become common and are rising at an alarming rate. Aminoacyl t-RNA synthetase has been validated as a newer target against Mycobacterium tuberculosis. Leucyl t-RNA synthetase (LeuRS) is ubiquitously found in all organisms and regulates transcription, protein synthesis, mitochondrial RNA cleavage, and proofreading of matured t-RNA. Leucyl t-RNA synthetase promotes growth and development and is the key enzyme needed for biofilm formation in Mycobacterium. Inhibition of this enzyme could restrict the growth and development of the mycobacterial population. A database consisting of 2734 drug-like molecules was screened against leucyl t-RNA synthetase enzymes through virtual screening. Based on the docking scores and MMGBSA energy values, the top three compounds were selected for molecular dynamics simulation. The druggable nature of the top three hits was confirmed by predicting their pharmacokinetic parameters. The top three hits—compounds 1035 (ZINC000001543916), 1054 (ZINC000001554197), and 2077 (ZINC000008214483)—were evaluated for their binding affinity toward leucyl t-RNA synthetase by an isothermal titration calorimetry study. The inhibitory activity of these compounds was tested against antimycobacterial activity, biofilm formation, and LeuRS gene expression potential. Compound 1054 (Macimorelin) was found to be the most potent molecule, with better antimycobacterial activity, enzyme binding affinity, and significant inhibition of biofilm formation, as well as inhibition of the LeuRS gene expression. Compound 1054, the top hit compound, has the potential to be used as a lead to develop successful leucyl t-RNA synthetase inhibitors. [ABSTRACT FROM AUTHOR]

Mitochondrial RNA modification (MRM) plays a crucial role in regulating the expression of key mitochondrial genes and promoting tumor metastasis. Despite its significance, comprehensive studies on MRM in lower grade gliomas (LGGs) remain unknown. Single-cell RNA-seq data (GSE89567) was used to evaluate the distribution functional status, and correlation of MRM-related genes in different cell types of LGG microenvironment. We developed an MRM scoring system by selecting potential MRM-related genes using LASSO regression analysis and the Random Survival Forest algorithm, based on multiple bulk RNA-seq datasets from TCGA, CGGA, GSE16011, and E-MTAB-3892. Analysis was performed on prognostic and immunological features, signaling pathways, metabolism, somatic mutations and copy number variations (CNVs), treatment responses, and forecasting of potential small-molecule agents. A total of 35 MRM-related genes were selected from the literature. Differential expression analysis of 1120 normal brain tissues and 529 LGGs revealed that 22 and 10 genes were upregulated and downregulated, respectively. Most genes were associated with prognosis of LGG. METLL8, METLL2A, TRMT112, and METTL2B were extensively expressed in all cell types and different cell cycle of each cell type. Almost all cell types had clusters related to mitochondrial RNA processing, ribosome biogenesis, or oxidative phosphorylation. Cell–cell communication and Pearson correlation analyses indicated that MRM may promoting the development of microenvironment beneficial to malignant progression via modulating NCMA signaling pathway and ICP expression. A total of 11 and 9 MRM-related genes were observed by LASSO and the RSF algorithm, respectively, and finally 6 MRM-related genes were used to establish MRM scoring system (TRMT2B, TRMT11, METTL6, METTL8, TRMT6, and TRUB2). The six MRM-related genes were then validated by qPCR in glioma and normal tissues. MRM score can predict the malignant clinical characteristics, abundance of immune infiltration, gene variation, clinical outcome, the enrichment of signaling pathways and metabolism. In vitro experiments demonstrated that silencing METTL8 significantly curbs glioma cell proliferation and enhances apoptosis. Patients with a high MRM score showed a better response to immunotherapies and small-molecule agents such as arachidonyl trifluoromethyl ketone, MS.275, AH.6809, tacrolimus, and TTNPB. These novel insights into the biological impacts of MRM within the glioma microenvironment underscore its potential as a target for developing precise therapies, including immunotherapeutic approaches. [ABSTRACT FROM AUTHOR]

Mitochondrial DNA replication is initiated by the transcription of mitochondrial RNA polymerase (mtRNAP), as mitochondria lack a dedicated primase. However, the mechanism determining the switch between continuous transcription and premature termination to generate RNA primers for mitochondrial DNA (mtDNA) replication remains unclear. The pentatricopeptide repeat domain of mtRNAP exhibits exoribonuclease activity, which is required for the initiation of mtDNA replication in Drosophila. In this review, we explain how this exonuclease activity contributes to primer synthesis in strand-coupled mtDNA replication, and discuss how its regulation might co-ordinate mtDNA replication and transcription in both Drosophila and mammals. [ABSTRACT FROM AUTHOR]

The heterogeneous composition of cellular transcriptomes poses a major challenge for detecting weakly expressed RNA classes, as they can be obscured by abundant RNAs. Although biochemical protocols can enrich or deplete specified RNAs, they are time-consuming, expensive and can compromise RNA integrity. Here we introduce RISER, a biochemical-free technology for the real-time enrichment or depletion of RNA classes. RISER performs selective rejection of molecules during direct RNA sequencing by identifying RNA classes directly from nanopore signals with deep learning and communicating with the sequencing hardware in real time. By targeting the dominant messenger and mitochondrial RNA classes for depletion, RISER reduces their respective read counts by more than 85%, resulting in an increase in sequencing depth of 47% on average for long non-coding RNAs. We also apply RISER for the depletion of globin mRNA in whole blood, achieving a decrease in globin reads by more than 90% as well as an increase in non-globin reads by 16% on average. Furthermore, using a GPU or a CPU, RISER is faster than GPU-accelerated basecalling and mapping. RISER's modular and retrainable software and intuitive command-line interface allow easy adaptation to other RNA classes. RISER is available at https://github.com/comprna/riser. It is difficult to detect low abundance RNAs in sequencing experiments, and biochemical methods to enrich or deplete specific RNAs are time-consuming, costly and can damage RNA. Here, authors develop a biochemical-free technology to enrich or deplete RNA classes in real-time during direct RNA sequencing. [ABSTRACT FROM AUTHOR]

Mitochondrial genomes of land plants are large and exhibit a complex mode of gene organization and expression, particularly at the post-transcriptional level. The primary organellar transcripts in plants undergo extensive maturation steps, including endo- and/or exo-nucleolytic cleavage, RNA-base modifications (mostly C-to-U deaminations) and both 'cis'- and 'trans'-splicing events. These essential processing steps rely on the activities of a large set of nuclear-encoded factors. RNA helicases serve as key players in RNA metabolism, participating in the regulation of transcription, mRNA processing and translation. They unwind RNA secondary structures and facilitate the formation of ribonucleoprotein complexes crucial for various stages of gene expression. Furthermore, RNA helicases are involved in RNA metabolism by modulating pre-mRNA maturation, transport and degradation processes. These enzymes are, therefore, pivotal in RNA quality-control mechanisms, ensuring the fidelity and efficiency of RNA processing and turnover in plant mitochondria. This review summarizes the significant roles played by helicases in regulating the highly dynamic processes of mitochondrial transcription, RNA processing and translation in plants. We further discuss recent advancements in understanding how dysregulation of mitochondrial RNA helicases affects the splicing of organellar genes, leading to respiratory dysfunctions, and consequently, altered growth, development and physiology of land plants. [ABSTRACT FROM AUTHOR]

Mitochondrial DNA gene organisation is an important source of phylogenetic information for various metazoan taxa at different evolutionary timescales, though this has not been broadly tested for all insect groups nor within a phylogenetic context. The cosmopolitan subfamily Doryctinae is a highly diverse group of braconid wasps mainly represented by ectoparasitoids of xylophagous beetle larvae. Previous molecular studies based on Sanger and genome-wide (ultraconserved elements, UCE; and mitochondrial genomes) sequence data have recovered a non-monophyletic Doryctinae, though the relationships involved have always been weakly supported. We characterised doryctine mitogenomes and conducted separate phylogenetic analyses based on mitogenome and UCE sequence data of ~100 representative doryctine genera to assess the monophyly and higher-level classification of the subfamily. We identified rearrangements of mitochondrial transfer RNAs (tRNAs) that support a non-monophyletic Doryctinae consisting of two separate non-related clades with strong geographic structure ('New World' and 'Old World' clades). This geographic structure was also consistently supported by the phylogenetic analyses preformed with mitogenome and UCE sequence data. These results highlight the utility of the mitogenome gene rearrangements as a potential source of phylogenetic information at different evolutionary timescales. Subfamily Doryctinae constitutes mainly parasitoid and some phytophagous braconid wasps. Molecular studies recovered a non-monophyletic Doryctinae. We characterised several mitogenomes and conducted phylogenetic analyses to assess the monophyly and higher-level classification. We identified rearrangements of mitochondrial transfer RNAs that support a non-monophyletic Doryctinae constituting two separate unrelated clades, also supported by phylogenetic analyses of mitogenome and nuclear data. Utility of the mitogenome rearrangements was highlighted as a potential source of phylogenetic information at different evolutionary timescales. (Image credit: Rubén Castañeda-Osorio.) [ABSTRACT FROM AUTHOR]

Significant variations in the abundance of mitochondrial RNA processing proteins and their target RNAs across trypanosome life stages present an opportunity to explore the regulatory mechanisms that drive these changes. Utilizing omics approaches can uncover unconventional targets, aiding our understanding of the parasites' adaptation and enabling targeted interventions for differentiation. [ABSTRACT FROM AUTHOR]

Defects in mitochondrial RNA metabolism have been linked to sensorineural deafness that often occurs as a consequence of damaged or deficient inner ear hair cells. In this report, we investigated the molecular mechanism underlying a deafnessassociated tRNAPhe 593T > C mutation that changed a highly conserved uracil to cytosine at position 17 of the DHU-loop. The m.593T > C mutation altered tRNAPhe structure and function, including increased melting temperature, resistance to S1 nuclease-mediated digestion, and conformational changes. The aberrant tRNA metabolism impaired mitochondrial translation, which was especially pronounced by decreases in levels of ND1, ND5, CYTB, CO1, and CO3 harboring higher numbers of phenylalanine. These alterations resulted in aberrant assembly, instability, and reduced activities of respiratory chain enzyme complexes I, III, IV, and intact supercomplexes overall. Furthermore, we found that the m.593T > C mutation caused markedly diminished membrane potential, and increased the production of reactive oxygen species in the mutant cell lines carrying the m.593T > C mutation. These mitochondrial dysfunctions led to the mitochondrial dynamic imbalance via increasing fission with abnormal mitochondrial morphology. Excessive fission impaired the process of autophagy including the initiation phase, formation, and maturation of the autophagosome. In particular, the m.593T > C mutation upregulated the PARKIN-dependent mitophagy pathway. These alterations promoted an intrinsic apoptotic process for the removal of damaged cells. Our findings provide critical insights into the pathophysiology of maternally inherited deafness arising from tRNA mutation-induced defects in mitochondrial and cellular integrity. [ABSTRACT FROM AUTHOR]

Kinetoplastid pathogens including Trypanosoma brucei, T. cruzi, and Leishmania species, are early diverged, eukaryotic, unicellular parasites. Functional understanding of many proteins from these pathogens has been hampered by limited sequence homology to proteins from other model organisms. Here we describe the development of a high-throughput deep mutational scanning approach in T. brucei that facilitates rapid and unbiased assessment of the impacts of many possible amino acid substitutions within a protein on cell fitness, as measured by relative cell growth. The approach leverages several molecular technologies: cells with conditional expression of a wild-type gene of interest and constitutive expression of a library of mutant variants, degroncontrolled stabilization of I-SceI meganuclease to mediate highly efficient transfection of a mutant allele library, and a high-throughput sequencing readout for cell growth upon conditional knockdown of wild-type gene expression and exclusive expression of mutant variants. Using this method, we queried the effects of amino acid substitutions in the apparently non-catalytic RNase III-like domain of KREPB4 (B4), which is an essential component of the RNA Editing Catalytic Complexes (RECCs) that carry out mitochondrial RNA editing in T. brucei. We measured the impacts of thousands of B4 variants on bloodstream form cell growth and validated the most deleterious variants containing single amino acid substitutions. Crucially, there was no correlation between phenotypes and amino acid conservation, demonstrating the greater power of this method over traditional sequence homology searching to identify functional residues. The bloodstream form cell growth phenotypes were combined with structural modeling, RECC protein proximity data, and analysis of selected substitutions in procyclic form T. brucei. These analyses revealed that the B4 RNaseIII-like domain is essential for maintenance of RECC integrity and RECC protein abundances and is also involved in changes in RECCs that occur between bloodstream and procyclic form life cycle stages. [ABSTRACT FROM AUTHOR]

Scutigeromorph centipedes are conspicuous, yet often ignored myriapods for which little work has been conducted in southern South America. After examining recent and museum collections from Chile and Argentina, two new species of generic uncertainty were identified. A new genus of scutigerid centipede, Edgethreua, is therefore described with two new species, E. chilensis from Central Chile (type species of the genus) and E. goloboffi from Argentinian Patagonia. The new genus is characterised by the presence of scattered setiform bristles with short paired spines and the absence of simple spinulae and spines on all stomatotergites, the presence of a single spine-bristle in the prefemur of the second maxilla, a patch of cuticular ridges and pores surrounding the sensilla of the proximal labral portion of the epipharynx, the morphology of the sensilla of the distal patch of the hypopharynx and the morphology of the female gonopods. A phylogenetic analysis of the new species using two nuclear ribosomal RNA genes (18S and 28S rRNA), two mitochondrial ribosomal RNA genes (12S and 16S rRNA) and the mitochondrial protein-encoding gene cytochrome c oxidase subunit I show that the new genus does not cluster with any other described genus of scutigeromorph represented in molecular phylogenies. The data indicate that the new genus is probably sister group to a clade including the genera Lassophora, Ballonema and the subfamily Thereuoneminae, although one analysis suggests a position as sister group to Scutigerinae. [ABSTRACT FROM AUTHOR]

Non-coding ribonucleic acids (ncRNAs) have been recently shown to contribute to tumorigenesis by mediating changes in metabolism. ncRNAs act as key molecules in metabolic pathways regulation. The dysregulation of ncRNAs during cancer progression contributes to altered metabolic phenotypes leading to reprogrammed metabolism. Since ncRNAs affect different tumor processes by regulating mitochondrial dynamics and metabolism, in the future ncRNAs can be exploited in disease detection, diagnosis, treatment, and resistance. The purpose of this review is to highlight the role of ncRNAs in mitochondrial metabolic reprogramming and to relate their therapeutic potential in the management of genitourinary cancer. [ABSTRACT FROM AUTHOR]

The mitochondrial genomes of apicomplexans comprise merely three protein-coding genes, alongside a set of thirty to forty genes encoding small RNAs (sRNAs), many of which exhibit homologies to rRNA from E. coli. The expression status and integration of these short RNAs into ribosomes remains unclear and direct evidence for active ribosomes within apicomplexan mitochondria is still lacking. In this study, we conducted small RNA sequencing on the apicomplexan Toxoplasma gondii to investigate the occurrence and function of mitochondrial sRNAs. To enhance the analysis of sRNA sequencing outcomes, we also re-sequenced the T. gondii mitochondrial genome using an improved organelle enrichment protocol and Nanopore sequencing. It has been established previously that the T. gondii genome comprises 21 sequence blocks that undergo recombination among themselves but that their order is not entirely random. The enhanced coverage of the mitochondrial genome allowed us to characterize block combinations at increased resolution. Employing this refined genome for sRNA mapping, we find that many small RNAs originated from the junction sites between protein-coding blocks and rRNA sequence blocks. Surprisingly, such block border sRNAs were incorporated into polysomes together with canonical rRNA fragments and mRNAs. In conclusion, apicomplexan ribosomes are active within polysomes and are indeed assembled through the integration of sRNAs, including previously undetected sRNAs with merged mRNA-rRNA sequences. Our findings lead to the hypothesis that T. gondii’s block-based genome organization enables the dual utilization of mitochondrial sequences as both messenger RNAs and ribosomal RNAs, potentially establishing a link between the regulation of rRNA and mRNA expression. [ABSTRACT FROM AUTHOR]

The significant role of long non-coding 7S RNA in controlling mitochondrial transcription highlights its importance in mitochondrial function. Considering the suggested connection between mitochondrial dysfunction and the onset of mental disorders, this study aimed to explore the potential involvement of 7S RNA in the context of depression/anxiety. A total of 181 patients in primary health care (age 20–64 years) with depression/anxiety and 59 healthy controls were included in the study. 7S RNA was measured using quantitative real-time PCR in plasma samples collected before (baseline) and after 8 weeks of treatment (mindfulness or cognitive-based behavioral therapy). Upon adjustment for age and sex, the baseline plasma levels of 7S RNA were significantly higher in patients than in healthy controls (p < 0.001). Notably, post-treatment, there was a significant reduction in 7S RNA levels (p = 0.03). These changes in 7S RNA were related to the treatment response, as indicated by HADS-D (Hospital Anxiety and Depression Scale) scores (ß = −0.04, p = 0.04), even after accounting for baseline scores and other cofounders. The findings of this study indicate an association between plasma 7S RNA levels and depression/anxiety, as well as treatment response. While further confirmatory analyses are necessary, plasma 7S RNA holds promise as a potential predictive biomarker for both depression/anxiety and the treatment response within these disorders. • Mitochondria non-coding RNA, 7S RNA regulates mitochondrial transcription. • The expression and the roles of 7S RNA in depression/anxiety remain unclear. • The initial expression levels of 7S RNA exhibited a notable increase in patients compared to the healthy control group. • Post-treatment, 7S RNA levels notably decreased, showing a connection with treatment response through HADS-D scores. [ABSTRACT FROM AUTHOR]

Cytidine (C)-to-uridine (U) RNA editing in plant organelles relies on specific RNA-binding pentatricopeptide repeat (PPR) proteins. In the moss Physcomitrium patens, all such RNA editing factors feature a C-terminal DYW domain that acts as the cytidine deaminase for C-to-U conversion. PPR78 of Physcomitrium targets 2 mitochondrial editing sites, cox1eU755SL and rps14eU137SL. Remarkably, the latter is edited to highly variable degrees in different mosses. Here, we aimed to unravel the coevolution of PPR78 and its 2 target sites in mosses. Heterologous complementation in a Physcomitrium knockout line revealed that the variable editing of rps14eU137SL depends on the PPR arrays of different PPR78 orthologues but not their C-terminal domains. Intriguingly, PPR78 has remained conserved despite the simultaneous loss of editing at both known targets among Hypnales (feather mosses), suggesting it serves an additional function. Using a recently established RNA editing assay in Escherichia coli, we confirmed site-specific RNA editing by PPR78 in the bacterium and identified 4 additional off-targets in the bacterial transcriptome. Based on conservation profiles, we predicted ccmFNeU1465RC as a candidate editing target of PPR78 in moss mitochondrial transcriptomes. We confirmed editing at this site in several mosses and verified that PPR78 targets ccmFNeU1465RC in the bacterial editing system, explaining the conservation and functional adaptation of PPR78 during moss evolution. The mitochondrial RNA editing factor PPR78 of Physcomitrium patens is conserved among mosses despite the loss of its 2 known C-to-U editing sites due to the additional editing target ccmFNeU1465RC. [ABSTRACT FROM AUTHOR]

A recent study published in Cell Biology International explores the potential of targeting mitochondrial RNA modifications as a therapeutic strategy to combat metastasis in late-stage cancers. The study focuses on the role of NSUN3, a mitochondrial RNA methyltransferase, and its involvement in the activation of tumor cell invasion and dissemination. The researchers found that inhibiting NSUN3-dependent RNA modifications can decrease the progression of lymph node metastasis without affecting primary tumor growth. Additionally, the study highlights the potential of antibiotics that inhibit mitochondrial translation as a treatment strategy to prevent cancer metastasis. Overall, this research provides valuable insights into the role of mitochondrial RNA modifications in tumor metastasis and suggests potential therapeutic targets for late-stage cancers. [Extracted from the article]

RNase P and RNase mitochondrial RNA processing (MRP) are ribonucleoproteins (RNPs) that consist of a catalytic RNA and a varying number of protein cofactors. RNase P is responsible for precursor tRNA maturation in all three domains of life, while RNase MRP, exclusive to eukaryotes, primarily functions in rRNA biogenesis. While eukaryotic RNase P is associated with more protein cofactors and has an RNA subunit with fewer auxiliary structural elements compared to its bacterial cousin, the double-anchor precursor tRNA recognition mechanism has remarkably been preserved during evolution. RNase MRP shares evolutionary and structural similarities with RNase P, preserving the catalytic core within the RNA moiety inherited from their common ancestor. By incorporating new protein cofactors and RNA elements, RNase MRP has established itself as a distinct RNP capable of processing ssRNA substrates. The structural information on RNase P and MRP helps build an evolutionary trajectory, depicting how emerging protein cofactors harmonize with the evolution of RNA to shape different functions for RNase P and MRP. Here, we outline the structural and functional relationship between RNase P and MRP to illustrate the coevolution of RNA and protein cofactors, a key driver for the extant, diverse RNP world. [ABSTRACT FROM AUTHOR]

Mood disorders, including major depressive disorder and bipolar disorder, are highly prevalent and stand among the leading causes of disability. Despite the largely elusive nature of the molecular mechanisms underpinning these disorders, two pivotal contributors--mitochondrial dysfunctions and epigenetic alterations--have emerged as significant players in their pathogenesis. This state-of-the-art review aims to present existing data on epigenetic alterations in the mitochondrial genome in mood disorders, laying the groundwork for future research into their pathogenesis. Associations between abnormalities in mitochondrial function and mood disorders have been observed, with evidence pointing to notable changes in mitochondrial DNA (mtDNA). These changes encompass variations in copy number and oxidative damage. However, information on additional epigenetic alterations in the mitochondrial genome remains limited. Recent studies have delved into alterations in mtDNA and regulations in the mitochondrial genome, giving rise to the burgeoning field of mitochondrial epigenetics. Mitochondrial epigenetics encompasses three main categories of modifications: mtDNA methylation/hydroxymethylation, modifications of mitochondrial nucleoids, and mitochondrial RNA alterations. The epigenetic modulation of mitochondrial nucleoids, lacking histones, may impact mtDNA function. Additionally, mitochondrial RNAs, including non-coding RNAs, present a complex landscape influencing interactions between the mitochondria and the nucleus. The exploration of mitochondrial epigenetics offers valuable perspectives on how these alterations impact neurodegenerative diseases, presenting an intriguing avenue for research on mood disorders. Investigations into post-translational modifications and the role of mitochondrial non-coding RNAs hold promise to unravel the dynamics of mitoepigenetics in mood disorders, providing crucial insights for future therapeutic interventions. [ABSTRACT FROM AUTHOR]

Simple Summary: The most common form of kidney cancer is clear cell renal cell carcinoma, but its treatment is challenging due to the acquisition of resistance to existing therapies. We propose an alternative potential treatment based on targeting a non-coding RNA of mitochondrial origin, termed antisense non-coding mitochondrial RNA. Supporting this strategy is the data we present in this work, using cell cultures obtained directly from primary and metastatic patient tumors, showing that the treatment causes a stall in cell proliferation in both in vitro and in vivo mouse models, and significantly improve the survival of animals. The results from this study strongly support the use of this strategy for the development of an alternative therapeutic approach for ccRCC patients and potentially other types of renal cancer. Clear cell renal cell carcinoma (ccRCC) is the most prevalent form of renal cancer and its treatment is hindered by a resistance to targeted therapies, immunotherapies and combinations of both. We have reported that the knockdown of the antisense noncoding mitochondrial RNAs (ASncmtRNAs) with chemically modified antisense oligonucleotides induces proliferative arrest and apoptotic death in tumor cells from many human and mouse cancer types. These studies have been mostly performed in vitro and in vivo on commercially available cancer cell lines and have shown that in mouse models tumor growth is stunted by the treatment. The present work was performed on cells derived from primary and metastatic ccRCC tumors. We established primary cultures from primary and metastatic ccRCC tumors, which were subjected to knockdown of ASncmtRNAs in vitro and in vivo in an orthotopic xenograft model in NOD/SCID mice. We found that these primary ccRCC cells are affected in the same way as tumor cell lines and in the orthotopic model tumor growth was significantly reduced by the treatment. This study on patient-derived ccRCC tumor cells represents a model closer to actual patient ccRCC tumors and shows that knockdown of ASncmtRNAs poses a potential treatment option for these patients. [ABSTRACT FROM AUTHOR]

Charcot-Marie-Tooth disease (CMT) is a group of inherited peripheral neuropathies related to variants in the mitochondrial transfer RNA (mt-tRNAval) gene. Here, we report a Chinese family harboring the m.1661A>G variant in the mt-tRNAval gene. Clinical evaluation, neuroelectrodiagnostic testing, and nerve biopsy were performed on four affected family members. Weakness, spasms, and pain in the limbs (especially in the lower limbs) were the main complaints of the proband. Physical examination revealed atrophy and weakness in the distal limbs, increased muscle tone, and hyperreflexia in four limbs. Neuroelectrodiagnostic tests and nerve biopsy supported an axonal polyneuropathy. This study furthers the understanding of phenotype diversity caused by variants in the mt-tRNAval gene in CMT. [ABSTRACT FROM AUTHOR]

The sub-compartmentalization of cellular processes is especially important in highly polarized cells such as neurons, as their function rely on their complex morphology. The association of RNAs to the mitochondrial surface is a conserved feature from yeast to humans and it regulates several aspects of mitochondrial physiology and, hence, cellular functions. In neurons, mitochondria are emerging as platforms for RNA transport and local protein translation. In this review, we discuss how RNA localization to mitochondria helps to sustain mitochondrial function, and how this can support mitochondrial homeostasis, especially in the distal parts of the neuron, to support neuronal activity. [ABSTRACT FROM AUTHOR]

Chromatin-associated RNAs (caRNAs) form a relatively poorly recognized layer of the epigenome. The caRNAs reported to date are transcribed from the nuclear genome. Here, leveraging a recently developed assay for detection of caRNAs and their genomic association, we report that mitochondrial RNAs (mtRNAs) are attached to the nuclear genome and constitute a subset of caRNA, thus termed mt-caRNA. In four human cell types analyzed, mt-caRNAs preferentially attach to promoter regions. In human endothelial cells (ECs), the level of mt-caRNA–promoter attachment changes in response to environmental stress that mimics diabetes. Suppression of a non-coding mt-caRNA in ECs attenuates stress-induced nascent RNA transcription from the nuclear genome, including that of critical genes regulating cell adhesion, and abolishes stress-induced monocyte adhesion, a hallmark of dysfunctional ECs. Finally, we report increased nuclear localization of multiple mtRNAs in the ECs of human diabetic donors, suggesting many mtRNA translocate to the nucleus in a cell stress and disease-dependent manner. These data nominate mt-caRNAs as messenger molecules responsible for mitochondrial–nuclear communication and connect the immediate product of mitochondrial transcription with the transcriptional regulation of the nuclear genome. [ABSTRACT FROM AUTHOR]

Background: Breast cancer (BC) has been linked to a variety of nuclear DNA changes as well as mitochondrial DNA (mtDNA) alterations. The study aims to evaluate/analyze the association of mitochondrial transfer RNA (mt-tRNA) leucine 2 gene with BC patients. Methods: In the current study, 24 samples have been collected from various families in Peshawar. DNA was extracted from blood. Polymerase chain reaction was used to amplify the mt-tRNA MT-TL2 gene, and 20 samples were sequenced. Results: The sequence was compared with accession #NC-012920.1 of the Revised Cambridge Reference Sequence (rCRS). The results (chromatograph, nucleotide sequence, and rCRS alignment) show mutations in mt-tRNA MT-TL2 gene in our participants is not the cause of BC. Conclusion: Yet, a significant number of BC patients must be studied, and their full mtDNA must be analyzed. This will provide an indication of the potential DNA marker that might be used to prevent BC deaths at the earliest stages. [ABSTRACT FROM AUTHOR]

Background The Asian leaf litter toads of the genus Leptobrachella Smith, 1925 (Anura, Megophryidae) inhabit the forest floor and rocky streams in hilly evergreen forests and are widely distributed from southern China, west to north-eastern India and Myanmar, through mainland Indochina to Peninsular Malaysia and the Island of Borneo. New information A new species of the Asian leaf litter toad genus Leptobrachella from Guizhou Province, China is described. Molecular phylogenetic analyses, based on mitochondrial 16S rRNA and COI genes and nuclear RAG1 gene sequences indicated that the new species is genetically divergent from its congeners. The new species could be distinguished from its congeners by a combination of the following characters: (1) body of medium size in males (SVL 31.9 - 32.9 mm); (2) distinct black spots present on flanks; (3) toes rudimentarily webbed, with wide lateral fringes; (4) skin on dorsum shagreened with fine tiny granules and short ridges; (5) heels overlapped when thighs are positioned at right angles to the body; (6) tibia-tarsal articulation reaching interior corner of the eye. A new species of the Asian leaf litter toad genus Leptobrachella from Guizhou Province, China is described. Molecular phylogenetic analyses, based on mitochondrial 16S rRNA and COI genes and nuclear RAG1 gene sequences indicated that the new species is genetically divergent from its congeners. The new species could be distinguished from its congeners by a combination of the following characters: (1) body of medium size in males (SVL 31.9 - 32.9 mm); (2) distinct black spots present on flanks; (3) toes rudimentarily webbed, with wide lateral fringes; (4) skin on dorsum shagreened with fine tiny granules and short ridges; (5) heels overlapped when thighs are positioned at right angles to the body; (6) tibia-tarsal articulation reaching interior corner of the eye. [ABSTRACT FROM AUTHOR]

The subfamily Rasborinae is a species-rich group of freshwater fish related to zebrafish; however, its taxonomy remains unclear. We present the complete mitogenome and corresponding polyadenylated mitotranscriptome of Rasbora rasbora (Hamilton 1822) based on long-read and high-coverage Oxford Nanopore Technology sequencing. The mitogenome size, gene content, and gene organization correspond to the typical vertebrate composition, and the mitogenome generates 10 polyadenylated mRNAs. Two alternative polyadenylation sites of ND5 mRNA were detected, one with a 596 nt 3′untranslated region corresponding to the antisense ND6 gene. Polyadenylation also generates seven of the mRNA UAA stop codons. Complete mitogenome sequences, excluding the control region, were carefully aligned for RNA-coding and protein-coding features using 54 available species of the subfamily Rasborinae. The phylogenetic analyses based on maximum likelihood, Bayesian inference, and neighbor-joining tree building methods confirm the transfer of R. rasbora into the Sumatrana species group. The overall phylogeny of the subfamily Rasborinae supports with high confidence some previously observed changes within this subfamily, as well as contradicts some conclusions set by previous studies.

Medulloblastoma is the most common malignant brain tumor in children. There are four groups, each with different causal mutations, affected pathways and prognosis. Here, we investigated the role of mitochondria in medulloblastoma and whether there are differences between the different groups. We compared the gene expression levels in the four different medulloblastoma groups (MB-WNT, MB-SHH, MB-G3 and MB-G4), with the focus on genes associated with mitochondria. We used several tools including Salmon, Tximeta, DESeq2, BiomaRt, STRING, Ggplot2, EnhancedVolcano, Venny 2.1 and Metscape. A total of 668 genes were differentially expressed and the most abundant genes were associated with cell division pathway followed by modulation of chemical synaptic transmission. We also identified several genes (ABAT, SOX9, ALDH5A, FOXM1, ABL1, NHLH1, NEUROD1 and NEUROD2) known to play vital role in medulloblastoma. Comparative expression analysis revealed OXPHOS complex-associated proteins of mitochondria. The most significantly expressed genes in the MB-SHH and MB-G4 groups were AHCYL1 and SFXN5 while PAICS was significantly upregulated in MB-WNT group. Notably, MB-G3 contained the most downregulated genes from the OXPHOS complexes, except COX6B2 which was strongly upregulated. We show the importance of mitochondria and compare their role in the four different medulloblastoma groups. [ABSTRACT FROM AUTHOR]

N6-methyladenosine (m6A) is the most prominent epitranscriptomic modification to RNA in eukaryotes, but it's role in adaptive changes within the gestational environment are poorly understood. We propose that gestational exposure to nano titanium dioxide (TiO2) contributes to cardiac m6A methylation in fetal offspring and influences mitochondrial gene expression. 10-week-old pregnant female FVB/NJ wild-type mice underwent 6 nonconsecutive days of whole-body inhalation exposure beginning on gestational day (GD) 5. Mice were exposed to filtered room air or nano-TiO2 with a target aerosol mass concentration of 12 mg/m3. At GD 15 mice were humanely killed and cardiac RNA and mitochondrial proteins extracted. Immunoprecipitation with m6A antibodies was performed followed by sequencing of immunoprecipitant (m6A) and input (mRNA) on the Illumina NextSeq 2000. Protein extraction, preparation, and LC-MS/MS were used for mitochondrial protein quantification. There were no differences in maternal or fetal pup weights, number of pups, or pup heart weights between exposure and control groups. Transcriptomic sequencing revealed 3648 differentially expressed mRNA in nano-TiO2 exposed mice (Padj ≤ 0.05). Transcripts involved in mitochondrial bioenergetics were significantly downregulated (83 of 85 genes). 921 transcripts revealed significant m6A methylation sites (Padj ≤ 0.10). 311 of the 921 mRNA were identified to have both 1) significantly altered expression and 2) differentially methylated sites. Mitochondrial proteomics revealed decreased expression of ATP Synthase subunits in the exposed group (P ≤ 0.05). The lack of m6A modifications to mitochondrial transcripts suggests a mechanism for decreased transcript stability and reduced protein expression due to gestational nano-TiO2 inhalation exposure. [ABSTRACT FROM AUTHOR]

Single cell RNAseq has been a big leap in many areas of biology. Rather than investigating gene expression on a whole organism level, this technology enables scientists to get a detailed look at rare single cells or within their cell population of interest. The field is growing, and many new methods appear each year. We compared methods utilized in our core facility: Smart-seq3, PlexWell, FLASH-seq, VASA-seq, SORT-seq, 10X, Evercode, and HIVE. We characterized the equipment requirements for each method. We evaluated the performances of these methods based on detected features, transcriptome diversity, mitochondrial RNA abundance and multiplets, among others and benchmarked them against bulk RNA sequencing. Here, we show that bulk transcriptome detects more unique transcripts than any single cell method. While most methods are comparable in many regards, FLASH-seq and VASA-seq yielded the best metrics, e.g., in number of features. If no equipment for automation is available or many cells are desired, then HIVE or 10X yield good results. In general, more recently developed methods perform better. This also leads to the conclusion that older methods should be phased out, and that the development of single cell RNAseq methods is still progressing considerably. [ABSTRACT FROM AUTHOR]

Background: Circulating extracellular vesicles (EVs) are increased in preeclampsia (PE) and are associated with severity and progression. We examined in this exploratory cohort study if the mRNAs and long noncoding RNAs (lncRNAs) in plasma-derived EVs were dysregulated in PE compared to normal pregnancy and display different temporal patterns during gestation. Methods: We isolated EVs from plasma at weeks 22–24 and 36–38 in women with and without PE (n=7 in each group) and performed RNA-seq, focusing on mRNAs and lncRNAs. We validated highly expressed mitochondrial and platelet-derived RNAs discovered from central pathways in 60 women with/without PE. We examined further one of the regulated RNAs, noncoding mitochondrially encoded tRNA alanine (MT-TA), in leukocytes and plasma to investigate its biomarker potential and association with clinical markers of PE. Results: We found abundant levels of platelet-derived and mitochondrial RNAs in EVs. Expression of these RNAs were decreased and lncRNAs increased in EVs from PE compared to without PE. These findings were further validated by qPCR for mitochondrial RNAs MT-TA, MT-ND2, MT-CYB and platelet-derived RNAs PPBP, PF4, CLU in EVs. Decreased expression of mitochondrial tRNA MT-TA in leukocytes at 22–24 weeks was strongly associated with the subsequent development of PE. Conclusions: Platelet-derived and mitochondrial RNA were highly expressed in plasma EVs and were decreased in EVs isolated from women with PE compared to without PE. LncRNAs were mostly increased in PE. The MT-TA in leukocytes may be a useful biomarker for prediction and/or early detection of PE. [ABSTRACT FROM AUTHOR]

SARS-CoV-2 causes the severe respiratory disease COVID-19. Remdesivir (RDV) was the first fast-tracked FDA approved treatment drug for COVID-19. RDV acts as an antiviral ribonucleoside (adenosine) analogue that becomes active once it accumulates intracellularly. It then diffuses into the host cell and terminates viral RNA transcription. Previous studies have shown that certain nucleoside analogues unintentionally inhibit mitochondrial RNA or DNA polymerases or cause mutational changes to mitochondrial DNA (mtDNA). These past findings on the mitochondrial toxicity of ribonucleoside analogues motivated us to investigate what effects RDV may have on mitochondrial function. Using in vitro and in vivo rodent models treated with RDV, we observed increases in mtDNA copy number in Mv1Lu cells (35.26% increase ± 11.33%) and liver (100.27% increase ± 32.73%) upon treatment. However, these increases only resulted in mild changes to mitochondrial function. Surprisingly, skeletal muscle and heart were extremely resistant to RDV treatment, tissues that have preferentially been affected by other nucleoside analogues. Although our data suggest that RDV does not greatly impact mitochondrial function, these data are insightful for the treatment of RDV for individuals with mitochondrial disease. [ABSTRACT FROM AUTHOR]

The mitochondrial proteome is subject to abundant post-translational modifications, including lysine acetylation and phosphorylation of serine, threonine, and tyrosine. The biological function of the majority of these protein modifications is unknown. Proteins required for the transcription and translation of mitochondrial DNA (mtDNA) are subject to modification. This suggests that reversible post-translational modifications may serve as a regulatory mechanism for mitochondrial gene transcription, akin to mechanisms controlling nuclear gene expression. We set out to determine whether acetylation or phosphorylation controls the function of mitochondrial RNA polymerase (POLRMT). Mass spectrometry was used to identify post-translational modifications on POLRMT. We analyzed three POLRMT modification sites (lysine 402, threonine 315, threonine 993) found in distinct structural regions. Amino acid point mutants that mimic the modified and unmodified forms of POLRMT were employed to measure the effect of acetylation or phosphorylation on the promoter binding ability of POLRMT in vitro. We found a slight decrease in binding affinity for the phosphomimic at threonine 315. We did not identify large changes in viability, mtDNA content, or mitochondrial transcript level upon overexpression of POLRMT modification mimics in HeLa cells. Our results suggest minimal biological impact of the POLRMT post-translational modifications studied in our system. [ABSTRACT FROM AUTHOR]

Simple Summary: Bladder cancer (BLCA) is one of the most prevalent urinary cancers and novel non-invasive BLCA tumor biomarkers with high sensitivity and specificity are needed to support early diagnosis. Exosomal long non-coding RNAs (lncRNAs) are promising diagnostic biomarkers for BLCA. Our study suggests that the combined evaluation of lncRNA RMRP levels in urinary and plasma exosomes is an excellent potential non-invasive diagnostic biomarker for BLCA patients. Additionally, targeting the RMRP/miR-206/G6PD axis is a promising strategy for BLCA treatment. Bladder cancer (BLCA) is one of the cancers that is highly sensitive to specific non-invasive tumor biomarkers that facilitate early diagnosis. Exosome-derived long non-coding RNAs (lncRNAs) hold promise as diagnostic biomarkers for BLCA. In this study, we employed RNA-sequencing to compare the expression patterns of lncRNAs in urine exosomes from three BLCA patients and three healthy individuals. RMRP displayed the most significant differential expression. Elevated RMRP expression levels were observed in urinary and plasma exosomes from BLCA patients compared with those from healthy individuals. RMRP exhibited significant associations with certain BLCA patient clinicopathological features, including tumor stage, poor prognosis, and tumor grade. Combined diagnosis using RMRP in urine and plasma exosomes demonstrated a superior diagnostic performance with receiver operating characteristic curve analysis. RMRP was found to be related to BLCA tumor progression and the cell migration and invasion processes via the miR-206/G6PD axis both in vitro and in vivo. Mechanistically, RMRP serves as an miR-206 sponge, as suggested by dual-luciferase reporter assays and RNA immunoprecipitation. Our study suggests that the combined diagnosis of RMRP in urinary and plasma exosomes can serve as an excellent non-invasive diagnostic biomarker for BLCA patients. Additionally, targeting the RMRP/miR-206/G6PD axis holds promise as a therapeutic strategy for BLCA. [ABSTRACT FROM AUTHOR]

Summary: Restorer‐of‐fertility (Rf) genes encode pentatricopeptide repeat (PPR) proteins that are targeted to mitochondria where they specifically bind to transcripts that induce cytoplasmic male sterility and repress their expression. In searching for a molecular signature unique to this class of proteins, we found that a majority of known Rf proteins have a distinct domain, which we called RfCTD (Restorer‐of‐fertility C‐terminal domain), and its presence correlates with the ability to induce cleavage of the mitochondrial RNA target.A screen of 219 angiosperm genomes from 123 genera using a sequence profile that can quickly and accurately identify RfCTD sequences revealed considerable variation in RFL/RfCTD gene numbers across flowering plants. We observed that plant genera with bisexual flowers have significantly higher numbers of RFL genes compared to those with unisexual flowers, consistent with a role of these proteins in restoration of male fertility.We show that removing the RfCTD from the RFL protein RNA PROCESSING FACTOR 2‐nad6 prevented cleavage of its RNA target, the nad6 transcript, in Arabidopsis thaliana mitochondria.We provide a simple way of identifying putative Rf candidates in genome sequences, new insights into the molecular mode of action of Rf proteins and the evolution of fertility restoration in flowering plants. [ABSTRACT FROM AUTHOR]

Summary: MicroRNAs (miRNAs) play crucial roles in regulating plant development and stress responses. However, the functions and mechanism of intronic miRNAs in plants are poorly understood.This study reports a stress‐responsive RNA splicing mechanism for intronic miR400 production, whereby miR400 modulates reactive oxygen species (ROS) accumulation and improves plant tolerance by downregulating its target expression.To monitor the intron splicing events, we used an intronic miR400 splicing‐dependent luciferase transgenic line. Luciferase activity was observed to decrease after high cadmium concentration treatment due to the retention of the miR400‐containing intron, which inhibited the production of mature miR400. Furthermore, we demonstrated that under Cd treatments, Pentatricopeptide Repeat Protein 1 (PPR1), the target of miR400, acts as a positive regulator by inducing ROS accumulation. Ppr1 mutation affected the Complex III activity in the electron transport chain and RNA editing of the mitochondrial gene ccmB.This study illustrates intron splicing as a key step in intronic miR400 production and highlights the function of intronic miRNAs as a 'signal transducer' in enhancing plant stress tolerance. [ABSTRACT FROM AUTHOR]

Although several pentatricopeptide repeat (PPR) proteins are involved in post-transcriptional processing of mitochondrial RNA, it is unclear which specific protein is involved in the RNA editing of ccmC in maize (Zea mays). Here we report the identification of the maize empty pericarp 601 (emp601) mutant and the map-based cloning of the Emp601 gene, which encodes an E2-type PPR protein that is targeted to mitochondria. A single-nucleotide deletion in the emp601 mutant caused a frameshift and introduced a premature stop codon into the predicted EMP601. This mutation was associated with reduced accumulation of mitochondrial complex III as well as with inhibition of growth and differentiation of basal endosperm transfer layer cells, leading to final degeneration of the embryo and endosperm. We determine that loss of EMP601 function prevents the C-to-U RNA editing of the mitochondrial transcript ccmC at position 358. EMP601 binds to the ccmC transcript and directly interacts with Multiple organellar RNA editing factor 8 and may be a component of the plant mitochondrial editosome. We conclude that EMP601 functions in RNA editing of mitochondrial ccmC transcripts and influences mitochondrial function and seed development. [ABSTRACT FROM AUTHOR]

Chromatin-associated RNAs (caRNAs) form a relatively poorly recognized layer of the epigenome. The caRNAs reported to date are transcribed from the nuclear genome. Here, leveraging a recently developed assay for detection of caRNAs and their genomic association, we report that mitochondrial RNAs (mtRNAs) are attached to the nuclear genome and constitute a subset of caRNA, thus termed mt-caRNA. In four human cell types analyzed, mt-caRNAs preferentially attach to promoter regions. In human endothelial cells (ECs), the level of mt-caRNA–promoter attachment changes in response to environmental stress that mimics diabetes. Suppression of a non-coding mt-caRNA in ECs attenuates stress-induced nascent RNA transcription from the nuclear genome, including that of critical genes regulating cell adhesion, and abolishes stress-induced monocyte adhesion, a hallmark of dysfunctional ECs. Finally, we report increased nuclear localization of multiple mtRNAs in the ECs of human diabetic donors, suggesting many mtRNA translocate to the nucleus in a cell stress and disease-dependent manner. These data nominate mt-caRNAs as messenger molecules responsible for mitochondrial–nuclear communication and connect the immediate product of mitochondrial transcription with the transcriptional regulation of the nuclear genome.