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Biological Chemistry Jul 2023Splicing of precursor mRNAs is a hallmark of eukaryotic cells, performed by a huge macromolecular machine, the spliceosome. Four DEAH-box ATPases are essential... (Review)
Review
Splicing of precursor mRNAs is a hallmark of eukaryotic cells, performed by a huge macromolecular machine, the spliceosome. Four DEAH-box ATPases are essential components of the spliceosome, which play an important role in the spliceosome activation, the splicing reaction, the release of the spliced mRNA and intron lariat, and the disassembly of the spliceosome. An integrative approach comprising X-ray crystallography, single particle cryo electron microscopy, single molecule FRET, and molecular dynamics simulations provided deep insights into the structure, dynamics and function of the spliceosomal DEAH-box ATPases.
Topics: Spliceosomes; Adenosine Triphosphatases; Saccharomyces cerevisiae Proteins; DEAD-box RNA Helicases; RNA Splicing
PubMed: 37441768
DOI: 10.1515/hsz-2023-0157 -
Journal of Hepatology Mar 2024In non-alcoholic fatty liver disease (NAFLD), monocytes infiltrate visceral adipose tissue promoting local and hepatic inflammation. However, it remains unclear what...
BACKGROUND & AIMS
In non-alcoholic fatty liver disease (NAFLD), monocytes infiltrate visceral adipose tissue promoting local and hepatic inflammation. However, it remains unclear what drives inflammation and how the immune landscape in adipose tissue differs across the NAFLD severity spectrum. We aimed to assess adipose tissue macrophage (ATM) heterogeneity in a NAFLD cohort.
METHODS
Visceral adipose tissue macrophages from lean and obese patients, stratified by NAFLD phenotypes, underwent single-cell RNA sequencing. Adipose tissue vascular integrity and breaching was assessed on a protein level via immunohistochemistry and immunofluorescence to determine targets of interest.
RESULTS
We discovered multiple ATM populations, including resident vasculature-associated macrophages (ResVAMs) and distinct metabolically active macrophages (MMacs). Using trajectory analysis, we show that ResVAMs and MMacs are replenished by a common transitional macrophage (TransMac) subtype and that, during NASH, MMacs are not effectively replenished by TransMac precursors. We postulate an accessory role for MMacs and ResVAMs in protecting the adipose tissue vascular barrier, since they both interact with endothelial cells and localize around the vasculature. However, across the NAFLD severity spectrum, alterations occur in these subsets that parallel an adipose tissue vasculature breach characterized by albumin extravasation into the perivascular tissue.
CONCLUSIONS
NAFLD-related macrophage dysfunction coincides with a loss of adipose tissue vascular integrity, providing a plausible mechanism by which tissue inflammation is perpetuated in adipose tissue and downstream in the liver.
IMPACT AND IMPLICATIONS
Our study describes for the first time the myeloid cell landscape in human visceral adipose tissue at single-cell level within a cohort of well-characterized patients with non-alcoholic fatty liver disease. We report unique non-alcoholic steatohepatitis-specific transcriptional changes within metabolically active macrophages (MMacs) and resident vasculature-associated macrophages (ResVAMs) and we demonstrate their spatial location surrounding the vasculature. These dysfunctional transcriptional macrophage states coincided with the loss of adipose tissue vascular integrity, providing a plausible mechanism by which tissue inflammation is perpetuated in adipose tissue and downstream in the liver. Our study provides a theoretical basis for new therapeutic strategies to be directed towards reinstating the endogenous metabolic, homeostatic and cytoprotective functions of ResVAMs and MMacs, including their role in protecting vascular integrity.
Topics: Humans; Non-alcoholic Fatty Liver Disease; Endothelial Cells; Liver; Macrophages; Adipose Tissue; Inflammation
PubMed: 37977244
DOI: 10.1016/j.jhep.2023.10.039 -
NAR Cancer Sep 2023Most cancer types exhibit aberrant transcriptional activity, including derepression of retrotransposable elements (RTEs). However, the degree, specificity and potential...
Most cancer types exhibit aberrant transcriptional activity, including derepression of retrotransposable elements (RTEs). However, the degree, specificity and potential consequences of RTE transcriptional activation may differ substantially among cancer types and subtypes. Representing one extreme of the spectrum, we characterize the transcriptional activity of RTEs in cohorts of esophageal adenocarcinoma (EAC) and its precursor Barrett's esophagus (BE) from the OCCAMS (Oesophageal Cancer Clinical and Molecular Stratification) consortium, and from TCGA (The Cancer Genome Atlas). We found exceptionally high RTE inclusion in the EAC transcriptome, driven primarily by transcription of genes incorporating intronic or adjacent RTEs, rather than by autonomous RTE transcription. Nevertheless, numerous chimeric transcripts straddling RTEs and genes, and transcripts from stand-alone RTEs, particularly KLF5- and SOX9-controlled proviruses, were overexpressed specifically in EAC. Notably, incomplete mRNA splicing and EAC-characteristic intronic RTE inclusion was mirrored by relative loss of the respective fully-spliced, functional mRNA isoforms, consistent with compromised cellular fitness. Defective RNA splicing was linked with strong transcriptional activation of a provirus on Chr Xp22.32 and defined EAC subtypes with distinct molecular features and prognosis. Our study defines distinguishable RTE transcriptional profiles of EAC, reflecting distinct underlying processes and prognosis, thus providing a framework for targeted studies.
PubMed: 37502711
DOI: 10.1093/narcan/zcad040 -
Circulation Research Mar 2024Chronic overconsumption of lipids followed by their excessive accumulation in the heart leads to cardiomyopathy. The cause of lipid-induced cardiomyopathy involves a...
BACKGROUND
Chronic overconsumption of lipids followed by their excessive accumulation in the heart leads to cardiomyopathy. The cause of lipid-induced cardiomyopathy involves a pivotal role for the proton-pump vacuolar-type H-ATPase (v-ATPase), which acidifies endosomes, and for lipid-transporter CD36, which is stored in acidified endosomes. During lipid overexposure, an increased influx of lipids into cardiomyocytes is sensed by v-ATPase, which then disassembles, causing endosomal de-acidification and expulsion of stored CD36 from the endosomes toward the sarcolemma. Once at the sarcolemma, CD36 not only increases lipid uptake but also interacts with inflammatory receptor TLR4 (Toll-like receptor 4), together resulting in lipid-induced insulin resistance, inflammation, fibrosis, and cardiac dysfunction. Strategies inducing v-ATPase reassembly, that is, to achieve CD36 reinternalization, may correct these maladaptive alterations. For this, we used NAD (nicotinamide adenine dinucleotide)-precursor nicotinamide mononucleotide (NMN), inducing v-ATPase reassembly by stimulating glycolytic enzymes to bind to v-ATPase.
METHODS
Rats/mice on cardiomyopathy-inducing high-fat diets were supplemented with NMN and for comparison with a cocktail of lysine/leucine/arginine (mTORC1 [mechanistic target of rapamycin complex 1]-mediated v-ATPase reassembly). We used the following methods: RNA sequencing, mRNA/protein expression analysis, immunofluorescence microscopy, (co)immunoprecipitation/proximity ligation assay (v-ATPase assembly), myocellular uptake of [H]chloroquine (endosomal pH), and [C]palmitate, targeted lipidomics, and echocardiography. To confirm the involvement of v-ATPase in the beneficial effects of both supplementations, mTORC1/v-ATPase inhibitors (rapamycin/bafilomycin A1) were administered. Additionally, 2 heart-specific v-ATPase-knockout mouse models (subunits VG1/Vd2) were subjected to these measurements. Mechanisms were confirmed in pharmacologically/genetically manipulated cardiomyocyte models of lipid overload.
RESULTS
NMN successfully preserved endosomal acidification during myocardial lipid overload by maintaining v-ATPase activity and subsequently prevented CD36-mediated lipid accumulation, CD36-TLR4 interaction toward inflammation, fibrosis, cardiac dysfunction, and whole-body insulin resistance. Lipidomics revealed C18:1-enriched diacylglycerols as lipid class prominently increased by high-fat diet and subsequently reversed/preserved by lysine/leucine/arginine/NMN treatment. Studies with mTORC1/v-ATPase inhibitors and heart-specific v-ATPase-knockout mice further confirmed the pivotal roles of v-ATPase in these beneficial actions.
CONCLUSION
NMN preserves heart function during lipid overload by preventing v-ATPase disassembly.
Topics: Animals; Mice; Rats; Adenosine Triphosphatases; Arginine; Cardiomyopathies; CD36 Antigens; Fibrosis; Inflammation; Insulin Resistance; Leucine; Lipids; Lysine; Mechanistic Target of Rapamycin Complex 1; Myocytes, Cardiac; Nicotinamide Mononucleotide; Toll-Like Receptor 4
PubMed: 38422177
DOI: 10.1161/CIRCRESAHA.123.322910 -
International Journal of Molecular... Sep 2023Splicing of pre-mRNA is a crucial regulatory stage in the pathway of gene expression. The majority of human genes that encode proteins undergo alternative pre-mRNA... (Review)
Review
Splicing of pre-mRNA is a crucial regulatory stage in the pathway of gene expression. The majority of human genes that encode proteins undergo alternative pre-mRNA splicing and mutations that affect splicing are more prevalent than previously thought. Targeting aberrant RNA(s) may thus provide an opportunity to correct faulty splicing and potentially treat numerous genetic disorders. To that purpose, the use of engineered U1 snRNA (either modified U1 snRNAs or exon-specific U1s-ExSpeU1s) has been applied as a potentially therapeutic strategy to correct splicing mutations, particularly those affecting the 5' splice-site (5'ss). Here we review and summarize a vast panoply of studies that used either modified U1 snRNAs or ExSpeU1s to mediate gene therapeutic correction of splicing defects underlying a considerable number of genetic diseases. We also focus on the pre-clinical validation of these therapeutic approaches both in vitro and in vivo, and summarize the main obstacles that need to be overcome to allow for their successful translation to clinic practice in the future.
Topics: Humans; RNA Precursors; RNA Splicing; RNA Splice Sites; RNA, Small Nuclear; Mutation; Alternative Splicing
PubMed: 37834063
DOI: 10.3390/ijms241914617 -
Genes Oct 2023Since the discovery of RNA splicing as a fundamental step to remove introns from pre-mRNA to produce mature mRNAs, substantial research in the past decades has...
Since the discovery of RNA splicing as a fundamental step to remove introns from pre-mRNA to produce mature mRNAs, substantial research in the past decades has highlighted RNA splicing as a critical mediator of gene expression and proteome diversity, also being important in many developmental and biological processes [...].
Topics: Humans; RNA Splicing; RNA Precursors; Neoplasms; RNA, Messenger; Introns
PubMed: 38002963
DOI: 10.3390/genes14112020 -
Nature Communications Aug 2023U6 snRNA is a catalytic RNA responsible for pre-mRNA splicing reactions and undergoes various post-transcriptional modifications during its maturation process. The...
U6 snRNA is a catalytic RNA responsible for pre-mRNA splicing reactions and undergoes various post-transcriptional modifications during its maturation process. The 3'-oligouridylation of U6 snRNA by the terminal uridylyltransferase, TUT1, provides the Lsm-binding site in U6 snRNA for U4/U6 di-snRNP formation and this ensures pre-mRNA splicing. Here, we present the crystal structure of human TUT1 (hTUT1) complexed with U6 snRNA, representing the post-uridylation of U6 snRNA by hTUT1. The N-terminal ZF-RRM and catalytic palm clamp the single-stranded AUA motif between the 5'-short stem and the 3'-telestem of U6 snRNA, and the ZF-RRM specifically recognizes the AUA motif. The ZF and the fingers hold the telestem, and the 3'-end of U6 snRNA is placed in the catalytic pocket of the palm for oligouridylation. The oligouridylation of U6 snRNA depends on the internal four-adenosine tract in the 5'-part of the telestem of U6 snRNA, and hTUT1 adds uridines until the internal adenosine tract can form base-pairs with the 3'-oligouridine tract. Together, the recognition of the specific structure and sequence of U6 snRNA by the multi-domain TUT1 protein and the intrinsic sequence and structure of U6 snRNA ensure the oligouridylation of U6 snRNA.
Topics: Humans; Adenosine; Nucleic Acid Conformation; Ribonucleoprotein, U4-U6 Small Nuclear; RNA Precursors; RNA Splicing; RNA, Small Nuclear
PubMed: 37563152
DOI: 10.1038/s41467-023-40420-9 -
Blood Advances Jul 2023The effect of aerobic glycolysis remains elusive in pediatric T-cell acute lymphoblastic leukemia (T-ALL). Increasing evidence has revealed that dysregulation of...
The effect of aerobic glycolysis remains elusive in pediatric T-cell acute lymphoblastic leukemia (T-ALL). Increasing evidence has revealed that dysregulation of deubiquitination is involved in glycolysis, by targeting glycolytic rate-limiting enzymes. Here, we demonstrated that upregulated deubiquitinase ubiquitin-specific peptidase 1 (USP1) expression correlated with poor prognosis in pediatric primary T-ALL samples. USP1 depletion abolished cellular proliferation and attenuated glycolytic metabolism. In vivo experiments showed that USP1 suppression decreased leukemia progression in nude mice. Inhibition of USP1 caused a decrease in both mRNA and protein levels in lactate dehydrogenase A (LDHA), a critical glycolytic enzyme. Moreover, USP1 interacted with and deubiquitinated polo-like kinase 1 (PLK1), a critical regulator of glycolysis. Overexpression of USP1 with upregulated PLK1 was observed in most samples of patients with T-ALL. In addition, PLK1 inhibition reduced LDHA expression and abrogated the USP1-mediated increase of cell proliferation and lactate level. Ectopic expression of LDHA can rescue the suppressive effect of USP1 silencing on cell growth and lactate production. Pharmacological inhibition of USP1 by ML323 exhibited cell cytotoxicity in human T-ALL cells. Taken together, our results demonstrated that USP1 may be a promising therapeutic target in pediatric T-ALL.
Topics: Animals; Child; Humans; Mice; Cell Line, Tumor; Disease Progression; Glycolysis; L-Lactate Dehydrogenase; Lactate Dehydrogenase 5; Lactates; Mice, Nude; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma; T-Lymphocytes; Ubiquitin-Specific Proteases; Polo-Like Kinase 1
PubMed: 36912760
DOI: 10.1182/bloodadvances.2022008284 -
Molecules (Basel, Switzerland) May 2024Azido-modified nucleosides have been extensively explored as substrates for click chemistry and the metabolic labeling of DNA and RNA. These compounds are also of... (Review)
Review
Azido-modified nucleosides have been extensively explored as substrates for click chemistry and the metabolic labeling of DNA and RNA. These compounds are also of interest as precursors for further synthetic elaboration and as therapeutic agents. This review discusses the chemistry of azidonucleosides related to the generation of nitrogen-centered radicals (NCRs) from the azido groups that are selectively inserted into the nucleoside frame along with the subsequent chemistry and biological implications of NCRs. For instance, the critical role of the sulfinylimine radical generated during inhibition of ribonucleotide reductases by 2'-azido-2'-deoxy pyrimidine nucleotides as well as the NCRs generated from azidonucleosides by radiation-produced (prehydrated and aqueous) electrons are discussed. Regio and stereoselectivity of incorporation of an azido group ("radical arm") into the frame of nucleoside and selective generation of NCRs under reductive conditions, which often produce the same radical species that are observed upon ionization events due to radiation and/or other oxidative conditions that are emphasized. NCRs generated from nucleoside-modified precursors other than azidonucleosides are also discussed but only with the direct relation to the same/similar NCRs derived from azidonucleosides.
Topics: Nucleosides; Azides; Nitrogen; Free Radicals; Click Chemistry
PubMed: 38792171
DOI: 10.3390/molecules29102310 -
Life Science Alliance Nov 2023Splicing factor 3B subunit 1 (SF3B1) is involved in pre-mRNA branch site recognition and is the target of antitumor-splicing inhibitors. Mutations in are observed in...
Splicing factor 3B subunit 1 (SF3B1) is involved in pre-mRNA branch site recognition and is the target of antitumor-splicing inhibitors. Mutations in are observed in 15% of patients with chronic lymphocytic leukemia (CLL) and are associated with poor prognosis, but their pathogenic mechanisms remain poorly understood. Using deep RNA-sequencing data from 298 CLL tumor samples and isogenic WT and K700E-mutated CLL cell lines, we characterize targets and pre-mRNA sequence features associated with the selection of cryptic 3' splice sites upon mutation, including an event in the gene relevant for activation of NF-κB signaling. Using the H3B-8800 splicing modulator, we show, for the first time in CLL, cytotoxic effects in vitro in primary CLL samples and in -mutated isogenic CLL cell lines, accompanied by major splicing changes and delayed leukemic infiltration in a CLL xenotransplant mouse model. H3B-8800 displayed preferential lethality towards -mutated cells and synergism with the BCL2 inhibitor venetoclax, supporting the potential use of SF3B1 inhibitors as a novel therapeutic strategy in CLL.
Topics: Mice; Animals; Leukemia, Lymphocytic, Chronic, B-Cell; RNA Splicing Factors; RNA Precursors; Phosphoproteins; Mutation; RNA Splice Sites; Antineoplastic Agents; Transcription Factors
PubMed: 37562845
DOI: 10.26508/lsa.202301955