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European Heart Journal May 2023Epicardium and epicardium-derived cells are critical players in myocardial fibrosis. Mesenchymal stem cell-derived extracellular vesicles (EVs) have been studied for...
AIMS
Epicardium and epicardium-derived cells are critical players in myocardial fibrosis. Mesenchymal stem cell-derived extracellular vesicles (EVs) have been studied for cardiac repair to improve cardiac remodelling, but the actual mechanisms remain elusive. The aim of this study is to investigate the mechanisms of EV therapy for improving cardiac remodelling and develop a promising treatment addressing myocardial fibrosis.
METHODS AND RESULTS
Extracellular vesicles were intrapericardially injected for mice myocardial infarction treatment. RNA-seq, in vitro gain- and loss-of-function experiments, and in vivo studies were performed to identify targets that can be used for myocardial fibrosis treatment. Afterward, a lipid nanoparticle-based long non-coding RNA (lncRNA) therapy was prepared for mouse and porcine models of myocardial infarction treatment. Intrapericardial injection of EVs improved adverse myocardial remodelling in mouse models of myocardial infarction. Mechanistically, Tcf21 was identified as a potential target to improve cardiac remodelling. Loss of Tcf21 function in epicardium-derived cells caused increased myofibroblast differentiation, whereas forced Tcf21 overexpression suppressed transforming growth factor-β signalling and myofibroblast differentiation. LncRNA-Tcf21 antisense RNA inducing demethylation (TARID) that enriched in EVs was identified to up-regulate Tcf21 expression. Formulated lncRNA-TARID-laden lipid nanoparticles up-regulated Tcf21 expression in epicardium-derived cells and improved cardiac function and histology in mouse and porcine models of myocardial infarction.
CONCLUSION
This study identified Tcf21 as a critical target for improving cardiac fibrosis. Up-regulating Tcf21 by using lncRNA-TARID-laden lipid nanoparticles could be a promising way to treat myocardial fibrosis. This study established novel mechanisms underlying EV therapy for improving adverse remodelling and proposed a lncRNA therapy for cardiac fibrosis.
Topics: Mice; Animals; Swine; RNA, Long Noncoding; RNA, Antisense; Ventricular Remodeling; Myocardial Infarction; Fibrosis; Demethylation
PubMed: 36916305
DOI: 10.1093/eurheartj/ehad114 -
RNA (New York, N.Y.) Mar 2020The use of synthetic RNA for therapeutics requires that the in vitro synthesis process be robust and efficient. The technology used for the synthesis of these in...
The use of synthetic RNA for therapeutics requires that the in vitro synthesis process be robust and efficient. The technology used for the synthesis of these in vitrotranscribed RNAs, predominantly using phage RNA polymerases (RNAPs), is well established. However, transcripts synthesized with RNAPs are known to display an immune-stimulatory activity in vivo that is often undesirable. Previous studies have identified double-stranded RNA (dsRNA), a major by-product of the in vitro transcription (IVT) process, as a trigger of cellular immune responses. Here we describe the characterization of a high-temperature IVT process using thermostable T7 RNAPs to synthesize functional mRNAs that demonstrate reduced immunogenicity without the need for a post-synthesis purification step. We identify features that drive the production of two kinds of dsRNA by-products-one arising from 3' extension of the run-off product and one formed by the production of antisense RNAs-and demonstrate that at a high temperature, T7 RNAP has reduced 3'-extension of the run-off product. We show that template-encoded poly(A) tailing does not affect 3'-extension but reduces the formation of the antisense RNA by-products. Combining high-temperature IVT with template-encoded poly(A) tailing prevents the formation of both kinds of dsRNA by-products generating functional mRNAs with reduced immunogenicity.
Topics: Bacteriophage T7; DNA-Directed RNA Polymerases; Immunity, Cellular; RNA; RNA, Antisense; RNA, Double-Stranded; RNA, Messenger; Transcription, Genetic
PubMed: 31900329
DOI: 10.1261/rna.073858.119 -
Science (New York, N.Y.) Dec 2008RNA polymerases are highly regulated molecular machines. We present a method (global run-on sequencing, GRO-seq) that maps the position, amount, and orientation of...
RNA polymerases are highly regulated molecular machines. We present a method (global run-on sequencing, GRO-seq) that maps the position, amount, and orientation of transcriptionally engaged RNA polymerases genome-wide. In this method, nuclear run-on RNA molecules are subjected to large-scale parallel sequencing and mapped to the genome. We show that peaks of promoter-proximal polymerase reside on approximately 30% of human genes, transcription extends beyond pre-messenger RNA 3' cleavage, and antisense transcription is prevalent. Additionally, most promoters have an engaged polymerase upstream and in an orientation opposite to the annotated gene. This divergent polymerase is associated with active genes but does not elongate effectively beyond the promoter. These results imply that the interplay between polymerases and regulators over broad promoter regions dictates the orientation and efficiency of productive transcription.
Topics: Cell Line; CpG Islands; DNA-Directed RNA Polymerases; Genome, Human; Humans; Nucleosomes; Promoter Regions, Genetic; RNA Polymerase II; RNA, Antisense; RNA, Messenger; Sequence Analysis, RNA; Transcription Initiation Site; Transcription, Genetic
PubMed: 19056941
DOI: 10.1126/science.1162228 -
Biochimie Nov 2011Although control of cellular function has classically been considered the responsibility of proteins, research over the last decade has elucidated many roles for RNA in... (Review)
Review
Although control of cellular function has classically been considered the responsibility of proteins, research over the last decade has elucidated many roles for RNA in regulation of not only the proteins that control cellular functions but also for the cellular functions themselves. In parallel to this advancement in knowledge about the regulatory roles of RNA there has been an explosion of knowledge about the role that epigenetics plays in controlling not only long-term cellular fate but also the short-term regulatory control of genes. Of particular interest is the crossover between these two worlds, a world where RNA can act out its part and subsequently elicit chromatin modifications that alter cellular function. Two main categories of RNA are examined here, non-coding RNA and antisense RNA both of which perform vital functions in controlling numerous genes, proteins and RNA itself. As the activities of non-coding and antisense RNA in both normal and aberrant cellular function are elucidated, so does the number of possible targets for pharmacopeic intervention.
Topics: Animals; Chromatin Assembly and Disassembly; Epigenesis, Genetic; Gene Expression Regulation; Genomic Imprinting; Humans; Pluripotent Stem Cells; RNA, Antisense; RNA, Untranslated; Transcription, Genetic; X Chromosome Inactivation
PubMed: 21843589
DOI: 10.1016/j.biochi.2011.07.031 -
RNA Biology 2014The symbiotic α-rhizobia Sinorhizobium meliloti, Bradyrhizobium japonicum, Rhizobium etli and the related plant pathogen Agrobacterium tumefaciens are important model... (Review)
Review
The symbiotic α-rhizobia Sinorhizobium meliloti, Bradyrhizobium japonicum, Rhizobium etli and the related plant pathogen Agrobacterium tumefaciens are important model organisms for studying plant-microbe interactions. These metabolically versatile soil bacteria are characterized by complex lifestyles and large genomes. Here we summarize the recent knowledge on their small non-coding RNAs (sRNAs) including conservation, function, and interaction of the sRNAs with the RNA chaperone Hfq. In each of these organisms, an inventory of hundreds of cis- and trans-encoded sRNAs with regulatory potential was uncovered by high-throughput approaches and used for the construction of 39 sRNA family models. Genome-wide analyses of hfq mutants and co-immunoprecipitation with tagged Hfq revealed a major impact of the RNA chaperone on the physiology of plant-associated α-proteobacteria including symbiosis and virulence. Highly conserved members of the SmelC411 family are the AbcR sRNAs, which predominantly regulate ABC transport systems. AbcR1 of A. tumefaciens controls the uptake of the plant-generated signaling molecule GABA and is a central regulator of nutrient uptake systems. It has similar functions in S. meliloti and the human pathogen Brucella abortus. As RNA degradation is an important process in RNA-based gene regulation, a short overview on ribonucleases in plant-associated α-proteobacteria concludes this review.
Topics: Alphaproteobacteria; Base Pairing; Gene Expression Regulation, Bacterial; Multigene Family; Plants; RNA Stability; RNA, Antisense; RNA, Bacterial; RNA, Messenger; RNA, Small Untranslated; RNA-Binding Proteins; Transcriptome
PubMed: 25003187
DOI: 10.4161/rna.29625 -
Microbiology Spectrum Jul 2018Although bacterial genomes are usually densely protein-coding, genome-wide mapping approaches of transcriptional start sites revealed that a significant fraction of the... (Review)
Review
Although bacterial genomes are usually densely protein-coding, genome-wide mapping approaches of transcriptional start sites revealed that a significant fraction of the identified promoters drive the transcription of noncoding RNAs. These can be -acting RNAs, mainly originating from intergenic regions and, in many studied examples, possessing regulatory functions. However, a significant fraction of these noncoding RNAs consist of natural antisense transcripts (asRNAs), which overlap other transcriptional units. Naturally occurring asRNAs were first observed to play a role in bacterial plasmid replication and in bacteriophage λ more than 30 years ago. Today's view is that asRNAs abound in all three domains of life. There are several examples of asRNAs in bacteria with clearly defined functions. Nevertheless, many asRNAs appear to result from pervasive initiation of transcription, and some data point toward global functions of such widespread transcriptional activity, explaining why the search for a specific regulatory role is sometimes futile. In this review, we give an overview about the occurrence of antisense transcription in bacteria, highlight particular examples of functionally characterized asRNAs, and discuss recent evidence pointing at global relevance in RNA processing and transcription-coupled DNA repair.
Topics: Bacteria; Bacterial Proteins; DNA Repair; Evolution, Molecular; Gene Expression Regulation, Bacterial; Genome, Bacterial; Plasmids; RNA, Antisense; RNA, Bacterial; RNA, Untranslated; Transcription, Genetic
PubMed: 30003872
DOI: 10.1128/microbiolspec.RWR-0029-2018 -
Human Molecular Genetics Sep 2014Recent years have seen the increasing understanding of the crucial role of RNA in the functioning of the eukaryotic genome. These discoveries, fueled by the achievements... (Review)
Review
Recent years have seen the increasing understanding of the crucial role of RNA in the functioning of the eukaryotic genome. These discoveries, fueled by the achievements of the FANTOM, and later GENCODE and ENCODE consortia, led to the recognition of the important regulatory roles of natural antisense transcripts (NATs) arising from what was previously thought to be 'junk DNA'. Roughly defined as non-coding regulatory RNA transcribed from the opposite strand of a coding gene locus, NATs are proving to be a heterogeneous group with high potential for therapeutic application. Here, we attempt to summarize the rapidly growing knowledge about this important non-coding RNA subclass.
Topics: Gene Expression; Gene Targeting; Genome; Humans; RNA, Antisense; RNA, Untranslated; Transcription, Genetic
PubMed: 24838284
DOI: 10.1093/hmg/ddu207 -
FEBS Letters Jun 2004Eukaryotes regulate gene expression in a number of different ways. On a daily and seasonal timescale, the orchestration of gene expression is to a large extent governed... (Review)
Review
Eukaryotes regulate gene expression in a number of different ways. On a daily and seasonal timescale, the orchestration of gene expression is to a large extent governed by circadian clocks. These endogenous timekeepers enable organisms to prepare for predictable environmental conditions from one day to the next and thus allow adaptation to a given temporal niche. In general, circadian clocks have been shown to employ the classical transcriptional and posttranscriptional control mechanisms to generate rhythmicity. However, the discovery of antisense clock gene transcripts suggests that mechanisms of gene regulation operating through antisense RNA may also be integral to the circadian clockwork. Following a brief history of the impact of genetic and molecular techniques in aiding our understanding of circadian clocks, this review concentrates on the few examples of antisense clock gene transcripts so far investigated and their effect on circadian timing.
Topics: Animals; Circadian Rhythm; Drosophila; Gene Expression Regulation; Models, Biological; Models, Genetic; Neurospora; Oligonucleotides, Antisense; RNA; RNA Processing, Post-Transcriptional; RNA, Antisense; RNA, Messenger; Time Factors
PubMed: 15165892
DOI: 10.1016/j.febslet.2004.04.073 -
Essays in Biochemistry Oct 2021RNA molecules have emerged as a new class of promising therapeutics to expand the range of druggable targets in the genome. In addition to 'canonical' protein-coding... (Review)
Review
RNA molecules have emerged as a new class of promising therapeutics to expand the range of druggable targets in the genome. In addition to 'canonical' protein-coding mRNAs, the emerging richness of sense and antisense long non-coding RNAs (lncRNAs) provides a new reservoir of molecular tools for RNA-based drugs. LncRNAs are composed of modular structural domains with specific activities involving the recruitment of protein cofactors or directly interacting with nucleic acids. A single therapeutic RNA transcript can then be assembled combining domains with defined secondary structures and functions, and antisense sequences specific for the RNA/DNA target of interest. As the first representative molecules of this new pharmacology, we have identified SINEUPs, a new functional class of natural antisense lncRNAs that increase the translation of partially overlapping mRNAs. Their activity is based on the combination of two domains: an embedded mouse inverted SINEB2 element that enhances mRNA translation (effector domain) and an overlapping antisense region that provides specificity for the target sense transcript (binding domain). By genetic engineering, synthetic SINEUPs can potentially target any mRNA of interest increasing translation and therefore the endogenous level of the encoded protein. In this review, we describe the state-of-the-art knowledge of SINEUPs and discuss recent publications showing their potential application in diseases where a physiological increase of endogenous protein expression can be therapeutic.
Topics: Animals; Mice; Protein Biosynthesis; Proteins; RNA, Antisense; RNA, Long Noncoding; RNA, Messenger
PubMed: 34623427
DOI: 10.1042/EBC20200114 -
Genes May 2021The leukocyte common antigen CD45 is a transmembrane phosphatase expressed on all nucleated hemopoietic cells, and the expression levels of its splicing isoforms are...
The leukocyte common antigen CD45 is a transmembrane phosphatase expressed on all nucleated hemopoietic cells, and the expression levels of its splicing isoforms are closely related to the development and function of lymphocytes. PEBP1P3 is a natural antisense transcript from the opposite strand of intron 2 and is predicted to be a noncoding RNA. The genotype-tissue expression and quantitative PCR data suggested that PEBP1P3 might be involved in the regulation of expression of CD45 splicing isoforms. To explore the regulatory mechanism of PEBP1P3 in CD45 expression, DNA methylation and histone modification were detected by bisulfate sequencing PCR and chromatin immunoprecipitation assays, respectively. The results showed that after the antisense RNA PEBP1P3 was knocked down by RNA interference, the DNA methylation of intron 2 was decreased and histone H3K9 and H3K36 trimethylation at the alternative splicing exons of DNA was increased. Knockdown of PEBP1P3 also increased the binding levels of chromatin conformation organizer CTCF at intron 2 and the alternative splicing exons of . The present results indicate that the natural antisense RNA PEBP1P3 regulated the alternative splicing of CD45 RNA, and that might be correlated with the regulation of histone modification and DNA methylation.
Topics: Alternative Splicing; CCCTC-Binding Factor; DNA Methylation; Histone Code; Humans; Jurkat Cells; Leukocyte Common Antigens; Protein Binding; Pseudogenes; RNA, Antisense
PubMed: 34067766
DOI: 10.3390/genes12050759