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Revealing the Arabidopsis AtGRP7 mRNA binding proteome by specific enhanced RNA interactome capture.BMC Plant Biology Jun 2024The interaction of proteins with RNA in the cell is crucial to orchestrate all steps of RNA processing. RNA interactome capture (RIC) techniques have been implemented to...
BACKGROUND
The interaction of proteins with RNA in the cell is crucial to orchestrate all steps of RNA processing. RNA interactome capture (RIC) techniques have been implemented to catalogue RNA- binding proteins in the cell. In RIC, RNA-protein complexes are stabilized by UV crosslinking in vivo. Polyadenylated RNAs and associated proteins are pulled down from cell lysates using oligo(dT) beads and the RNA-binding proteome is identified by quantitative mass spectrometry. However, insights into the RNA-binding proteome of a single RNA that would yield mechanistic information on how RNA expression patterns are orchestrated, are scarce.
RESULTS
Here, we explored RIC in Arabidopsis to identify proteins interacting with a single mRNA, using the circadian clock-regulated Arabidopsis thaliana GLYCINE-RICH RNA-BINDING PROTEIN 7 (AtGRP7) transcript, one of the most abundant transcripts in Arabidopsis, as a showcase. Seedlings were treated with UV light to covalently crosslink RNA and proteins. The AtGRP7 transcript was captured from cell lysates with antisense oligonucleotides directed against the 5'untranslated region (UTR). The efficiency of RNA capture was greatly improved by using locked nucleic acid (LNA)/DNA oligonucleotides, as done in the enhanced RIC protocol. Furthermore, performing a tandem capture with two rounds of pulldown with the 5'UTR oligonucleotide increased the yield. In total, we identified 356 proteins enriched relative to a pulldown from atgrp7 mutant plants. These were benchmarked against proteins pulled down from nuclear lysates by AtGRP7 in vitro transcripts immobilized on beads. Among the proteins validated by in vitro interaction we found the family of Acetylation Lowers Binding Affinity (ALBA) proteins. Interaction of ALBA4 with the AtGRP7 RNA was independently validated via individual-nucleotide resolution crosslinking and immunoprecipitation (iCLIP). The expression of the AtGRP7 transcript in an alba loss-of-function mutant was slightly changed compared to wild-type, demonstrating the functional relevance of the interaction.
CONCLUSION
We adapted specific RNA interactome capture with LNA/DNA oligonucleotides for use in plants using AtGRP7 as a showcase. We anticipate that with further optimization and up scaling the protocol should be applicable for less abundant transcripts.
Topics: Arabidopsis Proteins; Arabidopsis; RNA-Binding Proteins; Proteome; RNA, Messenger; RNA, Plant; Gene Expression Regulation, Plant
PubMed: 38877390
DOI: 10.1186/s12870-024-05249-4 -
Journal of Medical Case Reports Jun 2024Homozygous mutations in the APOA5 gene constitute a rare cause of monogenic hypertriglyceridemia, or familial chylomicronemia syndrome (FCS). We searched PubMed and...
BACKGROUND
Homozygous mutations in the APOA5 gene constitute a rare cause of monogenic hypertriglyceridemia, or familial chylomicronemia syndrome (FCS). We searched PubMed and identified 16 cases of homozygous mutations in the APOA5 gene. Severe hypertriglyceridemia related to monogenic mutations in triglyceride-regulating genes can cause recurrent acute pancreatitis. Standard therapeutic approaches for managing this condition typically include dietary interventions, fibrates, and omega-3-fatty acids. A novel therapeutic approach, antisense oligonucleotide volanesorsen is approved for use in patients with FCS.
CASE PRESENTATION
We report a case of a 25-years old Afghani male presenting with acute pancreatitis due to severe hypertriglyceridemia up to 29.8 mmol/L caused by homozygosity in APOA5 (c.427delC, p.Arg143Alafs*57). A low-fat diet enriched with medium-chain TG (MCT) oil and fibrate therapy did not prevent recurrent relapses, and volanesorsen was initiated. Volanesorsen resulted in almost normalized triglyceride levels. No further relapses of acute pancreatitis occurred. Patient reported an improve life quality due to alleviated chronic abdominal pain and headaches.
CONCLUSIONS
Our case reports a rare yet potentially life-threatening condition-monogenic hypertriglyceridemia-induced acute pancreatitis. The implementation of the antisense drug volanesorsen resulted in improved triglyceride levels, alleviated symptoms, and enhanced the quality of life.
Topics: Humans; Male; Adult; Pancreatitis; Apolipoprotein A-V; Hypertriglyceridemia; Recurrence; Homozygote; Mutation; Oligonucleotides; Hyperlipoproteinemia Type I; Diet, Fat-Restricted; Triglycerides
PubMed: 38872171
DOI: 10.1186/s13256-024-04532-0 -
Science Advances Jun 2024The function of TERRA in the regulation of telomerase in human cells is still debated. While TERRA interacts with telomerase, how it regulates telomerase function...
The function of TERRA in the regulation of telomerase in human cells is still debated. While TERRA interacts with telomerase, how it regulates telomerase function remains unknown. Here, we show that TERRA colocalizes with the telomerase RNA subunit hTR in the nucleoplasm and at telomeres during different phases of the cell cycle. We report that TERRA transcripts relocate away from chromosome ends during telomere lengthening, leading to a reduced number of telomeric TERRA-hTR molecules and consequent increase in "TERRA-free" telomerase molecules at telomeres. Using live-cell imaging and super-resolution microscopy, we show that upon transcription, TERRA relocates from its telomere of origin to long chromosome ends. Furthermore, TERRA depletion by antisense oligonucleotides promoted hTR localization to telomeres, leading to increased residence time and extended half-life of hTR molecules at telomeres. Overall, our findings indicate that telomeric TERRA transcripts inhibit telomere elongation by telomerase acting in trans, impairing telomerase access to telomeres that are different from their chromosome end of origin.
Topics: Telomerase; Humans; Telomere; Telomere Homeostasis; HeLa Cells; RNA; Transcription, Genetic; Telomere-Binding Proteins; Cell Cycle; Chromosomes, Human; DNA-Binding Proteins; Transcription Factors
PubMed: 38865460
DOI: 10.1126/sciadv.adk4387 -
Nature Communications Jun 2024Given the absence of approved treatments for pathogenic variants in Peripherin-2 (PRPH2), it is imperative to identify a universally effective therapeutic target for...
Given the absence of approved treatments for pathogenic variants in Peripherin-2 (PRPH2), it is imperative to identify a universally effective therapeutic target for PRPH2 pathogenic variants. To test the hypothesis that formation of the elongated discs in presence of PRPH2 pathogenic variants is due to the presence of the full complement of rhodopsin in absence of the required amounts of functional PRPH2. Here we demonstrate the therapeutic potential of reducing rhodopsin levels in ameliorating disease phenotype in knockin models for p.Lys154del (c.458-460del) and p.Tyr141Cys (c.422 A > G) in PRPH2. Reducing rhodopsin levels improves physiological function, mitigates the severity of disc abnormalities, and decreases retinal gliosis. Additionally, intravitreal injections of a rhodopsin-specific antisense oligonucleotide successfully enhance the physiological function of photoreceptors and improves the ultrastructure of discs in mutant mice. Presented findings shows that reducing rhodopsin levels is an effective therapeutic strategy for the treatment of inherited retinal degeneration associated with PRPH2 pathogenic variants.
Topics: Peripherins; Animals; Rhodopsin; Mice; Humans; Disease Models, Animal; Down-Regulation; Retinal Degeneration; Oligonucleotides, Antisense; Retina; Retinal Diseases; Mice, Inbred C57BL; Mutation; Female; Gene Knock-In Techniques; Male
PubMed: 38834544
DOI: 10.1038/s41467-024-48846-5 -
Frontiers in Pharmacology 2024Cardiovascular disease (CVD) poses a significant global health and economic challenge, with atherosclerosis being a primary cause. Over the past 40 years, substantial...
BACKGROUND
Cardiovascular disease (CVD) poses a significant global health and economic challenge, with atherosclerosis being a primary cause. Over the past 40 years, substantial research has been conducted into the prevention and reversal of atherosclerosis, resulting in the development of lipid-lowering agents such as statins and fibrates. Despite the extensive literature and formulation of numerous therapeutic guidelines in this domain, a comprehensive bibliometric analysis of the current research landscape and trends has not been performed. This study aimed to elucidate the evolution and milestones of research into lipid-lowering treatments for coronary heart disease (CHD) in conjunction with hyperlipidemia through bibliometric analysis, offering insights into future directions for treatment strategies.
METHODS
This study examined publications from 1986 to 2023 retrieved from the Web of Science database (Core Collection). Utilizing tools such as VOSviewer, Pajek, and CiteSpace, we analyzed publication and citation numbers, H-indexes, contributions by countries and institutions, authorship, journal sources, and keyword usage to uncover research trajectories and areas of focus.
RESULTS
Our analysis of 587 publications revealed a recent surge in research output, particularly post-2003. The American Journal of Cardiology published the highest number of studies, with 40 articles, whereas Circulation received the highest number of citations (6,266). Key contributors included the United States, Japan, and China, with the United States leading in citation numbers and the H-index. Harvard University and Leiden University emerged as pivotal institutions, and Professors J. Wouter Jukema and Robert P. Giugliano were identified as leading experts. Keyword analysis disclosed five thematic clusters, indicating a shift in research towards new drug combinations and strategies, signaling future research directions.
CONCLUSION
The last 4 decades have seen a notable rise in publications on lipid-lowering therapies for CHD and hyperlipidemia, with the United States retaining world-leading status. The increase in international collaboration aids the shift towards research into innovative lipid-lowering agents and therapeutic approaches. PCSK9 inhibitors and innovative combination therapies, including antisense oligonucleotides and angiopoietin-like protein 3 inhibitors, provide avenues for future research, intending to maximize the safety and efficacy of treatment approaches.
PubMed: 38828451
DOI: 10.3389/fphar.2024.1393333 -
Therapeutic Advances in Cardiovascular... 2024Cardiac fibrosis is a pivotal cardiovascular disease (CVD) process and represents a notable health concern worldwide. While the complex mechanisms underlying CVD have... (Review)
Review
Cardiac fibrosis is a pivotal cardiovascular disease (CVD) process and represents a notable health concern worldwide. While the complex mechanisms underlying CVD have been widely investigated, recent research has highlighted microRNA-21's (miR-21) role in cardiac fibrosis pathogenesis. In this narrative review, we explore the molecular interactions, focusing on the role of miR-21 in contributing to cardiac fibrosis. Various signaling pathways, such as the RAAS, TGF-β, IL-6, IL-1, ERK, PI3K-Akt, and PTEN pathways, besides dysregulation in fibroblast activity, matrix metalloproteinases (MMPs), and tissue inhibitors of MMPs cause cardiac fibrosis. Besides, miR-21 in growth factor secretion, apoptosis, and endothelial-to-mesenchymal transition play crucial roles. miR-21 capacity regulatory function presents promising insights for cardiac fibrosis. Moreover, this review discusses numerous approaches to control miR-21 expression, including antisense oligonucleotides, anti-miR-21 compounds, and Notch signaling modulation, all novel methods of cardiac fibrosis inhibition. In summary, this narrative review aims to assess the molecular mechanisms of cardiac fibrosis and its essential miR-21 function.
Topics: MicroRNAs; Humans; Fibrosis; Signal Transduction; Animals; Myocardium; Heart Diseases
PubMed: 38819836
DOI: 10.1177/17539447241253134 -
Molecular Therapy. Nucleic Acids Jun 2024
PubMed: 38803420
DOI: 10.1016/j.omtn.2024.102208 -
NAR Genomics and Bioinformatics Jun 2024Antisense oligonucleotides (ASOs) offer ground-breaking possibilities for selective pharmacological intervention for any gene product-related disease. Therapeutic ASOs...
Antisense oligonucleotides (ASOs) offer ground-breaking possibilities for selective pharmacological intervention for any gene product-related disease. Therapeutic ASOs contain extensive chemical modifications that improve stability to enzymatic cleavage and modulate binding affinity relative to natural RNA/DNA. Molecular dynamics (MD) simulation can provide valuable insights into how such modifications affect ASO conformational sampling and target binding. However, force field parameters for chemically modified nucleic acids (NAs) are still underdeveloped. To bridge this gap, we developed parameters to allow simulations of ASOs with the widely applied phosphorothioate (PS) backbone modification, and validated these in extensive all-atom MD simulations of relevant PS-modified NA systems representing B-DNA, RNA, and DNA/RNA hybrid duplex structures. Compared to the corresponding natural NAs, single PS substitutions had marginal effects on the ordered DNA/RNA duplex, whereas substantial effects of phosphorothioation were observed in single-stranded RNA and B-DNA, corroborated by the experimentally derived structure data. We find that PS-modified NAs shift between high and low twist states, which could affect target recognition and protein interactions for phosphorothioated oligonucleotides. Furthermore, conformational sampling was markedly altered in the PS-modified ssRNA system compared to that of the natural oligonucleotide, indicating sequence-dependent effects on conformational preference that may in turn influence duplex formation.
PubMed: 38800826
DOI: 10.1093/nargab/lqae058 -
ACS Central Science May 2024Thiol-mediated uptake (TMU) is an intriguing enigma in current chemistry and biology. While the appearance of cell-penetrating activity upon attachment of cascade...
Thiol-mediated uptake (TMU) is an intriguing enigma in current chemistry and biology. While the appearance of cell-penetrating activity upon attachment of cascade exchangers (CAXs) has been observed by many and is increasingly being used in practice, the molecular basis of TMU is essentially unknown. The objective of this study was to develop a general protocol to decode the dynamic covalent networks that presumably account for TMU. Uptake inhibition patterns obtained from the removal of exchange partners by either protein knockdown or alternative inhibitors are aligned with original patterns generated by CAX transporters and inhibitors and patterns from alternative functions (here cell motility). These inclusive TMU patterns reveal that the four most significant CAXs known today enter cells along three almost orthogonal pathways. Epidithiodiketopiperazines (ETP) exchange preferably with integrins and protein disulfide isomerases (PDIs), benzopolysulfanes (BPS) with different PDIs, presumably PDIA3, and asparagusic acid (AspA), and antisense oligonucleotide phosphorothioates (OPS) exchange with the transferrin receptor and can be activated by the removal of PDIs with their respective inhibitors. These findings provide a solid basis to understand and use TMU to enable and prevent entry into cells.
PubMed: 38799667
DOI: 10.1021/acscentsci.3c01601 -
STAR Protocols May 2024SCN2A loss-of-function variants cause a range of neurodevelopmental disorders. Here, we present a protocol to induce severe Scn2a insufficiency in mice. We describe...
SCN2A loss-of-function variants cause a range of neurodevelopmental disorders. Here, we present a protocol to induce severe Scn2a insufficiency in mice. We describe steps for intracerebroventricular (ICV) antisense oligonucleotide (ASO) injection that causes a selective downregulation of Scn2a and ASO-mediated mRNA degradation. We then detail procedures for qPCR and western blot protocol to measure Scn2a mRNA and protein. This protocol can be used as a mouse model for behavioral and in vivo two-photon Ca imaging.
PubMed: 38796847
DOI: 10.1016/j.xpro.2024.103094