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BioRxiv : the Preprint Server For... Feb 2024Pseudouridylation is a prevalent post-transcriptional RNA modification that impacts many aspects of RNA biology and function. The conversion of uridine to pseudouridine...
Pseudouridylation is a prevalent post-transcriptional RNA modification that impacts many aspects of RNA biology and function. The conversion of uridine to pseudouridine (Ψ) is catalyzed by the family of pseudouridine synthases (PUSs). Development of robust methods to determine PUS-dependent regulation of Ψ location and stoichiometry in low abundant mRNA is essential for biological and functional understanding of pseudouridylation. Here, we present a framework, NanoPsiPy, for identifying Ψ sites and quantify their levels in poly-A RNA at single-nucleotide resolution using direct RNA long-read Nanopore sequencing, based on the observation that Ψ can cause characteristic U-to-C basecalling errors in Nanopore direct RNA sequencing data. Our method was able to detect low and high stoichiometric Ψ sites in human mRNA. We validated our method by transcriptome-wide quantitative profiling of PUS7-dependent Ψ sites in poly-A RNA from a -amplified neuroblastoma cell line. We identified 8,625 PUS7-dependent Ψ sites in 1,246 mRNAs that encode proteins involved primarily in ribosome biogenesis, translation, and mitochondrial energy metabolism. Our work provides the first example of using direct RNA long-read Nanopore sequencing for transcriptome-wide quantitative profiling of mRNA pseudouridylation regulated by a PUS. We envision that our method will facilitate functional interrogation of PUSs in biological and pathological processes.
PubMed: 38352483
DOI: 10.1101/2024.01.31.578250 -
Plants (Basel, Switzerland) Jan 2024Erythromycin, a macrolide antibiotic, is a prioritized pollutant that poses a high risk to environmental health. It has been detected in different environmental matrices...
Erythromycin, a macrolide antibiotic, is a prioritized pollutant that poses a high risk to environmental health. It has been detected in different environmental matrices and can cause undesired effects in aquatic organisms, particularly freshwater algae, which are primary producers. However, the impact of erythromycin on marine algae remains largely unexplored. Erythromycin has been reported to induce hormetic effects in the marine diatom (). These effects are associated with the molecular pathways and biological processes of ribosome assembly, protein translation, photosynthesis, and oxidative stress. However, the alterations in the global gene expression have yet to be validated at the metabolic level. The present study used non-targeted metabolomic analysis to reveal the altered metabolic profiles of under erythromycin stress. The results showed that the increased cell density was possibly attributed to the accumulation of steroidal compounds with potential hormonic action at the metabolic level. Additionally, slight increases in the mitochondrial membrane potential (MMP) and viable cells were observed in the treatment of 0.001 mg/L of erythromycin (an environmentally realistic level). Contrarily, the 0.75 and 2.5 mg/L erythromycin treatments (corresponding to EC and EC, respectively) showed decreases in the MMP, cell density, and viable algal cells, which were associated with modified metabolic pathways involving ATP-binding cassette (ABC) transporters, the metabolism of hydrocarbons and lipids, thiamine metabolism, and the metabolism of porphyrin and chlorophyll. These findings suggest that metabolomic analysis, as a complement to the measurement of apical endpoints, could provide novel insights into the molecular mechanisms of hormesis induced by antibiotic agents in algae.
PubMed: 38337887
DOI: 10.3390/plants13030354 -
The Journal of Biological Chemistry Mar 2024Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by dysregulation of the expression and processing of the amyloid precursor protein...
Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by dysregulation of the expression and processing of the amyloid precursor protein (APP). Protein quality control systems are dedicated to remove faulty and deleterious proteins to maintain cellular protein homeostasis (proteostasis). Identidying mechanisms underlying APP protein regulation is crucial for understanding AD pathogenesis. However, the factors and associated molecular mechanisms regulating APP protein quality control remain poorly defined. In this study, we show that mutant APP with its mitochondrial-targeting sequence ablated exhibited predominant endoplasmic reticulum (ER) distribution and led to aberrant ER morphology, deficits in locomotor activity, and shortened lifespan. We searched for regulators that could counteract the toxicity caused by the ectopic expression of this mutant APP. Genetic removal of the ribosome-associated quality control (RQC) factor RACK1 resulted in reduced levels of ectopically expressed mutant APP. By contrast, gain of RACK1 function increased mutant APP level. Additionally, overexpression of the ER stress regulator (IRE1) resulted in reduced levels of ectopically expressed mutant APP. Mechanistically, the RQC related ATPase VCP/p97 and the E3 ubiquitin ligase Hrd1 were required for the reduction of mutant APP level by IRE1. These factors also regulated the expression and toxicity of ectopically expressed wild type APP, supporting their relevance to APP biology. Our results reveal functions of RACK1 and IRE1 in regulating the quality control of APP homeostasis and mitigating its pathogenic effects, with implications for the understanding and treatment of AD.
Topics: Animals; Alzheimer Disease; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Drosophila Proteins; Protein Serine-Threonine Kinases; Receptors for Activated C Kinase; Drosophila melanogaster; Disease Models, Animal; Endoribonucleases
PubMed: 38311171
DOI: 10.1016/j.jbc.2024.105719 -
Aging Jan 2024Mitochondrial ribosomal protein S23 (MRPS23), a component of the ribosome small subunit, has been reported to be overexpressed in various cancers and has been predicted...
Mitochondrial ribosomal protein S23 (MRPS23), a component of the ribosome small subunit, has been reported to be overexpressed in various cancers and has been predicted to be involved in increased cell proliferation. It has been confirmed that MRPS23 was involved in the regulation of breast cancer and hepatocellular carcinoma cell proliferation. However, little is known about the function of MRPS23 in glioma. In this study, we found that MRPS23 expression was higher in gliomas than in adjacent normal tissues. Higher expression of MRPS23 in gliomas correlated with poorer prognosis, unfavorable histological features, absence of mutations in the isocitrate dehydrogenase gene (IDH), absence of chromosome 1p and 19q deletions, and responses to chemoradiotherapy. Univariate and multivariate Cox analysis demonstrated MRPS23 expression was independently prognostic of overall survival, disease-free survival, and progression-free survival in patients with glioma. KEGG enrichment analysis results indicated that high MRPS23 expression was associated with cell proliferation and immune response-related signaling pathways. We also confirmed that MRPS23 was highly expressed in glioma cells lines, and MRPS23 knockdown significantly reduced cell survival, proliferation, and migration of glioma cells lines. Collectively, these findings offer mechanistic insights into how MRPS23 during glioma progression, and identify MRPS23 as a potential therapeutic target in the future.
Topics: Humans; Prognosis; Brain Neoplasms; Biomarkers, Tumor; Glioma; Mutation; Isocitrate Dehydrogenase
PubMed: 38301044
DOI: 10.18632/aging.205493 -
Circulation Research Feb 2024Human cardiac long noncoding RNA (lncRNA) profiles in patients with dilated cardiomyopathy (DCM) were previously analyzed, and the long noncoding RNA CHKB (choline...
BACKGROUND
Human cardiac long noncoding RNA (lncRNA) profiles in patients with dilated cardiomyopathy (DCM) were previously analyzed, and the long noncoding RNA CHKB (choline kinase beta) divergent transcript (CHKB-DT) levels were found to be mostly downregulated in the heart. In this study, the function of CHKB-DT in DCM was determined.
METHODS
Long noncoding RNA expression levels in the human heart tissues were measured via quantitative reverse transcription-polymerase chain reaction and in situ hybridization assays. A CHKB-DT heterozygous or homozygous knockout mouse model was generated using the clustered regularly interspaced palindromic repeat (CRISPR)/CRISPR-associated protein 9 system, and the adeno-associated virus with a cardiac-specific promoter was used to deliver the RNA in vivo. Sarcomere shortening was performed to assess the primary cardiomyocyte contractility. The Seahorse XF cell mitochondrial stress test was performed to determine the energy metabolism and ATP production. Furthermore, the underlying mechanisms were explored using quantitative proteomics, ribosome profiling, RNA antisense purification assays, mass spectrometry, RNA pull-down, luciferase assay, RNA-fluorescence in situ hybridization, and Western blotting.
RESULTS
CHKB-DT levels were remarkably decreased in patients with DCM and mice with transverse aortic constriction-induced heart failure. Heterozygous knockout of CHKB-DT in cardiomyocytes caused cardiac dilation and dysfunction and reduced the contractility of primary cardiomyocytes. Moreover, CHKB-DT heterozygous knockout impaired mitochondrial function and decreased ATP production as well as cardiac energy metabolism. Mechanistically, ALDH2 (aldehyde dehydrogenase 2) was a direct target of CHKB-DT. CHKB-DT physically interacted with the mRNA of ALDH2 and fused in sarcoma (FUS) through the GGUG motif. CHKB-DT knockdown aggravated ALDH2 mRNA degradation and 4-HNE (4-hydroxy-2-nonenal) production, whereas overexpression of CHKB-DT reversed these molecular changes. Furthermore, restoring ALDH2 expression in CHKB-DT mice alleviated cardiac dilation and dysfunction.
CONCLUSIONS
CHKB-DT is significantly downregulated in DCM. CHKB-DT acts as an energy metabolism-associated long noncoding RNA and represents a promising therapeutic target against DCM.
Topics: Animals; Humans; Mice; Adenosine Triphosphate; Aldehyde Dehydrogenase, Mitochondrial; Cardiomyopathy, Dilated; Down-Regulation; In Situ Hybridization, Fluorescence; Mice, Knockout; Mitochondria, Heart; Myocytes, Cardiac; RNA, Long Noncoding
PubMed: 38299365
DOI: 10.1161/CIRCRESAHA.123.323428 -
Cell Discovery Jan 2024Mitochondrial rRNA modifications are essential for mitoribosome assembly and its proper function. The mC methyltransferase METTL15 maintains mitochondrial homeostasis by...
Mitochondrial rRNA modifications are essential for mitoribosome assembly and its proper function. The mC methyltransferase METTL15 maintains mitochondrial homeostasis by catalyzing mC839 located in 12 S rRNA helix 44 (h44). This modification is essential to fine-tuning the ribosomal decoding center and increasing decoding fidelity according to studies of a conserved site in Escherichia coli. Here, we reported a series of crystal structures of human METTL15-hsRBFA-h44-SAM analog, METTL15-hsRBFA-SAM, METTL15-SAM and apo METTL15. The structures presented specific interactions of METTL15 with different substrates and revealed that hsRBFA recruits METTL15 to mitochondrial small subunit for further modification instead of 12 S rRNA. Finally, we found that METTL15 deficiency caused increased reactive oxygen species, decreased membrane potential and altered cellular metabolic state. Knocking down METTL15 caused an elevated lactate secretion and increased levels of histone H4K12-lactylation and H3K9-lactylation. METTL15 might be a suitable model to study the regulation between mitochondrial metabolism and histone lactylation.
PubMed: 38291322
DOI: 10.1038/s41421-023-00634-z -
Acta Neuropathologica Jan 2024The prevalence of epilepsy is increased among Alzheimer's Disease (AD) patients and cognitive impairment is common among people with epilepsy. Epilepsy and AD are linked...
The prevalence of epilepsy is increased among Alzheimer's Disease (AD) patients and cognitive impairment is common among people with epilepsy. Epilepsy and AD are linked but the shared pathophysiological changes remain poorly defined. We aim to identify protein differences associated with epilepsy and AD using published proteomics datasets. We observed a highly significant overlap in protein differences in epilepsy and AD: 89% (689/777) of proteins altered in the hippocampus of epilepsy patients were significantly altered in advanced AD. Of the proteins altered in both epilepsy and AD, 340 were altered in the same direction, while 216 proteins were altered in the opposite direction. Synapse and mitochondrial proteins were markedly decreased in epilepsy and AD, suggesting common disease mechanisms. In contrast, ribosome proteins were increased in epilepsy but decreased in AD. Notably, many of the proteins altered in epilepsy interact with tau or are regulated by tau expression. This suggests that tau likely mediates common protein changes in epilepsy and AD. Immunohistochemistry for Aβ and multiple phosphorylated tau species (pTau396/404, pTau217, pTau231) showed a trend for increased intraneuronal pTau217 and pTau231 but no phosphorylated tau aggregates or amyloid plaques in epilepsy hippocampal sections. Our results provide insights into common mechanisms in epilepsy and AD and highlights the potential role of tau in mediating common pathological protein changes in epilepsy and AD.
Topics: Humans; Alzheimer Disease; Proteomics; Brain; Epilepsy; Ribosomal Proteins
PubMed: 38289539
DOI: 10.1007/s00401-024-02683-4 -
Human Molecular Genetics May 2024Human mitochondrial DNA is one of the most simplified cellular genomes and facilitates compartmentalized gene expression. Within the organelle, there is no physical... (Review)
Review
Human mitochondrial DNA is one of the most simplified cellular genomes and facilitates compartmentalized gene expression. Within the organelle, there is no physical barrier to separate transcription and translation, nor is there evidence that quality control surveillance pathways are active to prevent translation on faulty mRNA transcripts. Mitochondrial ribosomes synthesize 13 hydrophobic proteins that require co-translational insertion into the inner membrane of the organelle. To maintain the integrity of the inner membrane, which is essential for organelle function, requires responsive quality control mechanisms to recognize aberrations in protein synthesis. In this review, we explore how defects in mitochondrial protein synthesis can arise due to the culmination of inherent mistakes that occur throughout the steps of gene expression. In turn, we examine the stepwise series of quality control processes that are needed to eliminate any mistakes that would perturb organelle homeostasis. We aim to provide an integrated view on the quality control mechanisms of mitochondrial protein synthesis and to identify promising avenues for future research.
Topics: Humans; Protein Biosynthesis; Mitochondrial Proteins; Mitochondria; DNA, Mitochondrial; RNA, Messenger; Mitochondrial Ribosomes; Animals
PubMed: 38280230
DOI: 10.1093/hmg/ddae012 -
Clinical Epigenetics Jan 2024Periodontitis is a highly prevalent oral disease characterized by bacterium-induced periodontal inflammation and alveolar bone destruction. Osteoblast function is...
BACKGROUND
Periodontitis is a highly prevalent oral disease characterized by bacterium-induced periodontal inflammation and alveolar bone destruction. Osteoblast function is impaired in periodontitis with a global proteome change. METTL3 is the pivotal methyltransferase of N-methyladenosine (mA) that is recently proved to exert a crucial role in osteoblast differentiation. This study aims to investigate the role of METTL3 in osteoblast ribosome biogenesis in periodontitis progression.
RESULTS
METTL3 was knocked down in osteoblasts, and the downregulated genes were enriched in ribosome and translation. METTL3 knockdown inhibited ribosome biogenesis and oxidative phosphorylation in LPS-stimulated osteoblasts, whereas METTL3 overexpression facilitated ribosomal and mitochondrial function. Mechanistically, METTL3 mediated osteoblast biological behaviors by activating Wnt/β-catenin/c-Myc signaling. METTL3 depletion enhanced the mRNA expression and stability of Dkk3 and Sostdc1 via YTHDF2. In periodontitis mice, METTL3 inhibitor SAH promoted alveolar bone loss and local inflammatory status, which were partially rescued by Wnt/β-catenin pathway activator CHIR-99021 HCl.
CONCLUSIONS
METTL3 promoted ribosome biogenesis and oxidative phosphorylation by activating Wnt/β-catenin/c-Myc signaling in LPS-treated osteoblasts and alleviated the inflammatory alveolar bone destruction in periodontitis mice.
Topics: Animals; Mice; beta Catenin; DNA Methylation; Lipopolysaccharides; Methyltransferases; Osteoblasts; Periodontitis; Proto-Oncogene Proteins c-myc; Signal Transduction
PubMed: 38267969
DOI: 10.1186/s13148-024-01628-8 -
Frontiers in Physiology 2023Osteoporosis (OP) is a chronic bone metabolic disease and a serious global public health problem. Several studies have shown that mitophagy plays an important role in...
Osteoporosis (OP) is a chronic bone metabolic disease and a serious global public health problem. Several studies have shown that mitophagy plays an important role in bone metabolism disorders; however, its role in osteoporosis remains unclear. The Gene Expression Omnibus (GEO) database was used to download GSE56815, a dataset containing low and high BMD, and differentially expressed genes (DEGs) were analyzed. Mitochondrial autophagy-related genes (MRG) were downloaded from the existing literature, and highly correlated MRG were screened by bioinformatics methods. The results from both were taken as differentially expressed (DE)-MRG, and Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were performed. Protein-protein interaction network (PPI) analysis, support vector machine recursive feature elimination (SVM-RFE), and Boruta method were used to identify DE-MRG. A receiver operating characteristic curve (ROC) was drawn, a nomogram model was constructed to determine its diagnostic value, and a variety of bioinformatics methods were used to verify the relationship between these related genes and OP, including GO and KEGG analysis, IP pathway analysis, and single-sample Gene Set Enrichment Analysis (ssGSEA). In addition, a hub gene-related network was constructed and potential drugs for the treatment of OP were predicted. Finally, the specific genes were verified by real-time quantitative polymerase chain reaction (RT-qPCR). In total, 548 DEGs were identified in the GSE56815 dataset. The weighted gene co-expression network analysis(WGCNA) identified 2291 key module genes, and 91 DE-MRG were obtained by combining the two. The PPI network revealed that the target gene for AKT1 interacted with most proteins. Three MRG (NELFB, SFSWAP, and MAP3K3) were identified as hub genes, with areas under the curve (AUC) 0.75, 0.71, and 0.70, respectively. The nomogram model has high diagnostic value. GO and KEGG analysis showed that ribosome pathway and cellular ribosome pathway may be the pathways regulating the progression of OP. IPA showed that MAP3K3 was associated with six pathways, including GNRH Signaling. The ssGSEA indicated that NELFB was highly correlated with iDCs (cor = -0.390, < 0.001). The regulatory network showed a complex relationship between miRNA, transcription factor(TF) and hub genes. In addition, 4 drugs such as vinclozolin were predicted to be potential therapeutic drugs for OP. In RT-qPCR verification, the hub gene NELFB was consistent with the results of bioinformatics analysis. Mitophagy plays an important role in the development of osteoporosis. The identification of three mitophagy-related genes may contribute to the early diagnosis, mechanism research and treatment of OP.
PubMed: 38260098
DOI: 10.3389/fphys.2023.1289976