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Biomolecules Aug 2023Dystrophinopathies are x-linked muscular disorders which emerge from mutations in the gene, including Duchenne and Becker muscular dystrophy, and dilated... (Review)
Review
Dystrophinopathies are x-linked muscular disorders which emerge from mutations in the gene, including Duchenne and Becker muscular dystrophy, and dilated cardiomyopathy. However, Duchenne muscular dystrophy interconnects with bone loss and osteoporosis, which are exacerbated by glucocorticoids therapy. Procedures for diagnosing dystrophinopathies include creatine kinase assay, haplotype analysis, Southern blot analysis, immunological analysis, multiplex PCR, multiplex ligation-dependent probe amplification, Sanger DNA sequencing, and next generation DNA sequencing. Pharmacological therapy for dystrophinopathies comprises glucocorticoids (prednisone, prednisolone, and deflazacort), vamorolone, and ataluren. However, angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers (ARBs), and β-blockers are the first-line to prevent dilated cardiomyopathy in dystrophinopathy patients. Duchenne muscular dystrophy gene therapy strategies involve gene transfer, exon skipping, exon reframing, and CRISPR gene editing. Eteplirsen, an antisense-oligonucleotide drug for skipping exon 51 from the gene, is available on the market, which may help up to 14% of Duchenne muscular dystrophy patients. There are various FDA-approved exon skipping drugs including ExonDys-51 for exon 51, VyonDys-53 and Viltolarsen for exon 53 and AmonDys-45 for exon 45 skipping. Other antisense oligonucleotide drugs in the pipeline include casimersen for exon 45, suvodirsen for exon 51, and golodirsen for exon 53 skipping. Advances in the diagnosis and therapy of dystrophinopathies offer new perspectives for their early discovery and care.
Topics: Humans; Dystrophin; Muscular Dystrophy, Duchenne; Cardiomyopathy, Dilated; Angiotensin Receptor Antagonists; Angiotensin-Converting Enzyme Inhibitors; Oligonucleotides, Antisense
PubMed: 37759719
DOI: 10.3390/biom13091319 -
Autophagy Jan 2024AKI: acute kidney injury; ATP: adenosine triphosphate; BUN: blood urea nitrogen; CLP: cecal ligation and puncture; eGFR: estimated glomerular filtration rate; H&E:...
AKI: acute kidney injury; ATP: adenosine triphosphate; BUN: blood urea nitrogen; CLP: cecal ligation and puncture; eGFR: estimated glomerular filtration rate; H&E: hematoxylin and eosin staining; LCN2/NGAL: lipocalin 2; LPS: lipopolysaccharide; LTL: lotus tetragonolobus lectin; mKeima: mitochondria-targeted Keima; mtDNA: mitochondrial DNA; PAS: periodic acid - Schiff staining; RTECs: renal tubular epithelial cells; SAKI: sepsis-induced acute kidney injury; Scr: serum creatinine; SIRT3: sirtuin 3; TFAM: transcription factor A, mitochondrial; TMRE: tetramethylrhodamine.
Topics: Humans; Sirtuin 3; Melatonin; Mitophagy; Autophagy; Acute Kidney Injury; Lipopolysaccharides; DNA, Mitochondrial; Sepsis; Kidney; DNA-Binding Proteins; Transcription Factors; Mitochondrial Proteins
PubMed: 37651673
DOI: 10.1080/15548627.2023.2252265 -
Nature Biotechnology Apr 2024Little is known about the biological roles of glycosylated RNAs (glycoRNAs), a recently discovered class of glycosylated molecules, because of a lack of visualization...
Little is known about the biological roles of glycosylated RNAs (glycoRNAs), a recently discovered class of glycosylated molecules, because of a lack of visualization methods. We report sialic acid aptamer and RNA in situ hybridization-mediated proximity ligation assay (ARPLA) to visualize glycoRNAs in single cells with high sensitivity and selectivity. The signal output of ARPLA occurs only when dual recognition of a glycan and an RNA triggers in situ ligation, followed by rolling circle amplification of a complementary DNA, which generates a fluorescent signal by binding fluorophore-labeled oligonucleotides. Using ARPLA, we detect spatial distributions of glycoRNAs on the cell surface and their colocalization with lipid rafts as well as the intracellular trafficking of glycoRNAs through SNARE protein-mediated secretory exocytosis. Studies in breast cell lines suggest that surface glycoRNA is inversely associated with tumor malignancy and metastasis. Investigation of the relationship between glycoRNAs and monocyte-endothelial cell interactions suggests that glycoRNAs may mediate cell-cell interactions during the immune response.
Topics: Cell Line; Oligonucleotides; RNA
PubMed: 37217750
DOI: 10.1038/s41587-023-01801-z -
BioRxiv : the Preprint Server For... Feb 2024In biology, DNA is often tightly bent to small radii. Solely based on the groove asymmetry, a 30-year-old theoretical paper predicted that such bending should unwind...
In biology, DNA is often tightly bent to small radii. Solely based on the groove asymmetry, a 30-year-old theoretical paper predicted that such bending should unwind DNA, but this effect has not been directly experimentally quantified so far. We developed a ligation-based assay with nicked DNA circles of variable length, thereby decoupling the twist-dependent ligation efficiency from the large bending strain which dominates conventional circularization assays. We demonstrate that tightly bent DNA indeed unwinds to over 11 base pairs/turn, exactly as predicted. Our discovery requires reassessing the molecular mechanisms and energetics of all processes where DNA is tightly bent or relaxed again, including DNA packaging, gene regulation and expression.
PubMed: 38405957
DOI: 10.1101/2024.02.14.579968 -
Nature Mar 2024Cyclic GMP-AMP synthase (cGAS) senses aberrant DNA during infection, cancer and inflammatory disease, and initiates potent innate immune responses through the synthesis...
Cyclic GMP-AMP synthase (cGAS) senses aberrant DNA during infection, cancer and inflammatory disease, and initiates potent innate immune responses through the synthesis of 2'3'-cyclic GMP-AMP (cGAMP). The indiscriminate activity of cGAS towards DNA demands tight regulatory mechanisms that are necessary to maintain cell and tissue homeostasis under normal conditions. Inside the cell nucleus, anchoring to nucleosomes and competition with chromatin architectural proteins jointly prohibit cGAS activation by genomic DNA. However, the fate of nuclear cGAS and its role in cell physiology remains unclear. Here we show that the ubiquitin proteasomal system (UPS) degrades nuclear cGAS in cycling cells. We identify SPSB3 as the cGAS-targeting substrate receptor that associates with the cullin-RING ubiquitin ligase 5 (CRL5) complex to ligate ubiquitin onto nuclear cGAS. A cryo-electron microscopy structure of nucleosome-bound cGAS in a complex with SPSB3 reveals a highly conserved Asn-Asn (NN) minimal degron motif at the C terminus of cGAS that directs SPSB3 recruitment, ubiquitylation and cGAS protein stability. Interference with SPSB3-regulated nuclear cGAS degradation primes cells for type I interferon signalling, conferring heightened protection against infection by DNA viruses. Our research defines protein degradation as a determinant of cGAS regulation in the nucleus and provides structural insights into an element of cGAS that is amenable to therapeutic exploitation.
Topics: Animals; Humans; Mice; Cell Nucleus; Cryoelectron Microscopy; Degrons; DNA Virus Infections; DNA Viruses; DNA, Viral; Immunity, Innate; Innate Immunity Recognition; Interferon Type I; Nuclear Proteins; Nucleosomes; Nucleotidyltransferases; Proteasome Endopeptidase Complex; Protein Stability; Proteolysis; Substrate Specificity; Ubiquitin; Ubiquitin-Protein Ligases; Ubiquitination
PubMed: 38418882
DOI: 10.1038/s41586-024-07112-w -
Biochemical Society Transactions Oct 2023Non-homologous end joining (NHEJ) is the major pathway for the repair of ionizing radiation-induced DNA double-strand breaks (DSBs) in human cells and is essential for... (Review)
Review
Non-homologous end joining (NHEJ) is the major pathway for the repair of ionizing radiation-induced DNA double-strand breaks (DSBs) in human cells and is essential for the generation of mature T and B cells in the adaptive immune system via the process of V(D)J recombination. Here, we review how recently determined structures shed light on how NHEJ complexes function at DNA DSBs, emphasizing how multiple structures containing the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) may function in NHEJ. Together, these studies provide an explanation for how NHEJ proteins assemble to detect and protect DSB ends, then proceed, through DNA-PKcs-dependent autophosphorylation, to a ligation-competent complex.
Topics: Humans; DNA-Binding Proteins; DNA End-Joining Repair; DNA Breaks, Double-Stranded; Phosphorylation; DNA; DNA Repair
PubMed: 37787023
DOI: 10.1042/BST20220741 -
BioRxiv : the Preprint Server For... Oct 2023Oncogene amplification on extrachromosomal DNA (ecDNA) is a pervasive driver event in cancer, yet our understanding of how ecDNA forms is limited. Here, we couple a...
Oncogene amplification on extrachromosomal DNA (ecDNA) is a pervasive driver event in cancer, yet our understanding of how ecDNA forms is limited. Here, we couple a CRISPR-based method for induction of ecDNA with extensive characterization of newly formed ecDNA to examine ecDNA biogenesis. We find that DNA circularization is efficient, irrespective of 3D genome context, with formation of a 1 Mb and 1.8 Mb ecDNA both reaching 15%. We show non-homologous end joining and microhomology mediated end joining both contribute to ecDNA formation, while inhibition of DNA-PKcs and ATM have opposing impacts on ecDNA formation. EcDNA and the corresponding chromosomal excision scar form at significantly different rates and respond differently to DNA-PKcs and ATM inhibition. Taken together, our results support a model of ecDNA formation in which double strand break ends dissociate from their legitimate ligation partners prior to joining of illegitimate ends to form the ecDNA and excision scar.
PubMed: 37961138
DOI: 10.1101/2023.10.22.563489 -
Genome Biology Oct 2023Genomic abnormalities are strongly associated with cancer and infertility. In this study, we develop a simple and efficient method - multiple genetic abnormality...
Genomic abnormalities are strongly associated with cancer and infertility. In this study, we develop a simple and efficient method - multiple genetic abnormality sequencing (MGA-Seq) - to simultaneously detect structural variation, copy number variation, single-nucleotide polymorphism, homogeneously staining regions, and extrachromosomal DNA (ecDNA) from a single tube. MGA-Seq directly sequences proximity-ligated genomic fragments, yielding a dataset with concurrent genome three-dimensional and whole-genome sequencing information, enabling approximate localization of genomic structural variations and facilitating breakpoint identification. Additionally, by utilizing MGA-Seq, we map focal amplification and oncogene coamplification, thus facilitating the exploration of ecDNA's transcriptional regulatory function.
Topics: DNA Copy Number Variations; Oncogenes; Genomics; Gene Expression Regulation; DNA
PubMed: 37904244
DOI: 10.1186/s13059-023-03081-x -
Genes & Development Jul 2023Punctuated bursts of structural genomic variations (SVs) have been described in various organisms, but their etiology remains incompletely understood. Homologous...
Punctuated bursts of structural genomic variations (SVs) have been described in various organisms, but their etiology remains incompletely understood. Homologous recombination (HR) is a template-guided mechanism of repair of DNA double-strand breaks and stalled or collapsed replication forks. We recently identified a DNA break amplification and genome rearrangement pathway originating from the endonucleolytic processing of a multi-invasion (MI) DNA joint molecule formed during HR. Genome-wide approaches confirmed that multi-invasion-induced rearrangement (MIR) frequently leads to several repeat-mediated SVs and aneuploidies. Using molecular and genetic analysis and a novel, highly sensitive proximity ligation-based assay for chromosomal rearrangement quantification, we further delineate two MIR subpathways. MIR1 is a universal pathway occurring in any sequence context, which generates secondary breaks and frequently leads to additional SVs. MIR2 occurs only if recombining donors exhibit substantial homology and results in sequence insertion without additional breaks or SVs. The most detrimental MIR1 pathway occurs late on a subset of persisting DNA joint molecules in a PCNA/Pol-independent manner, unlike recombinational DNA synthesis. This work provides a refined mechanistic understanding of these HR-based SV formation pathways and shows that complex repeat-mediated SVs can occur without displacement DNA synthesis. Sequence signatures for inferring MIR1 from long-read data are proposed.
Topics: Genomic Instability; Gene Rearrangement; Homologous Recombination; Selection, Genetic; DNA; Chromosomes, Fungal; Saccharomyces cerevisiae
PubMed: 37541760
DOI: 10.1101/gad.350618.123