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Ageing Research Reviews Sep 2012Senescence represents a state of indefinite growth arrest in cells that have reached the end of their replicative life span, have become damaged, or express aberrant... (Review)
Review
Senescence represents a state of indefinite growth arrest in cells that have reached the end of their replicative life span, have become damaged, or express aberrant levels of cancer-related proteins. While senescence is widely considered to represent a tumor-suppressive mechanism, the accumulation of senescent cells in tissues of older organisms is believed to underlie age-associated losses in physiologic function and age-related diseases. With the emergence of microRNAs (miRNAs) as a major class of molecular regulators of senescence, we review the transcriptional and post-transcriptional factors that control senescence-associated microRNA biosynthesis. Focusing on their enhancement or repression of senescence, we describe the transcription factors that govern the synthesis of primary (pri-)miRNAs, the proteins that control the nuclear processing of pri-miRNAs into precursor (pre-)miRNAs, including RNA editing enzymes, RNases, and RNA helicases, and the cytoplasmic proteins that affect the final processing of pre-miRNAs into mature miRNAs. We discuss how miRNA biogenesis proteins promote or inhibit senescence, and thus influence the senescent phenotype that affects normal tissue function and pathology.
Topics: Animals; Cellular Senescence; Gene Expression Regulation; Humans; MicroRNAs; RNA Editing; RNA Processing, Post-Transcriptional; RNA-Binding Proteins
PubMed: 22306790
DOI: 10.1016/j.arr.2012.01.003 -
Genome Research Sep 2015Precise regulation of microRNA (miRNA) expression is critical for diverse physiologic and pathophysiologic processes. Nevertheless, elucidation of the mechanisms through...
Precise regulation of microRNA (miRNA) expression is critical for diverse physiologic and pathophysiologic processes. Nevertheless, elucidation of the mechanisms through which miRNA expression is regulated has been greatly hindered by the incomplete annotation of primary miRNA (pri-miRNA) transcripts. While a subset of miRNAs are hosted in protein-coding genes, the majority of pri-miRNAs are transcribed as poorly characterized noncoding RNAs that are 10's to 100's of kilobases in length and low in abundance due to efficient processing by the endoribonuclease DROSHA, which initiates miRNA biogenesis. Accordingly, these transcripts are poorly represented in existing RNA-seq data sets and exhibit limited and inaccurate annotation in current transcriptome assemblies. To overcome these challenges, we developed an experimental and computational approach that allows genome-wide detection and mapping of pri-miRNA structures. Deep RNA-seq in cells expressing dominant-negative DROSHA resulted in much greater coverage of pri-miRNA transcripts compared with standard RNA-seq. A computational pipeline was developed that produces highly accurate pri-miRNA assemblies, as confirmed by extensive validation. This approach was applied to a panel of human and mouse cell lines, providing pri-miRNA transcript structures for 1291/1871 human and 888/1181 mouse miRNAs, including 594 human and 425 mouse miRNAs that fall outside protein-coding genes. These new assemblies uncovered unanticipated features and new potential regulatory mechanisms, including links between pri-miRNAs and distant protein-coding genes, alternative pri-miRNA splicing, and transcripts carrying subsets of miRNAs encoded by polycistronic clusters. These results dramatically expand our understanding of the organization of miRNA-encoding genes and provide a valuable resource for the study of mammalian miRNA regulation.
Topics: Alternative Splicing; Evolution, Molecular; Gene Expression Regulation; Genome-Wide Association Study; High-Throughput Nucleotide Sequencing; Humans; MicroRNAs; Molecular Sequence Annotation; Multigene Family; Nucleic Acid Conformation; Open Reading Frames; Promoter Regions, Genetic; RNA, Untranslated; Ribonuclease III; Sequence Analysis, RNA; Transcription, Genetic
PubMed: 26290535
DOI: 10.1101/gr.193607.115 -
Viruses Jan 2023Virotherapy is a promising, novel form of cancer immunotherapy currently being investigated in pre-clinical and clinical settings. While generally well-tolerated, the...
Virotherapy is a promising, novel form of cancer immunotherapy currently being investigated in pre-clinical and clinical settings. While generally well-tolerated, the anti-tumor potency of oncolytic virus-based monotherapies needs to be improved further. One of the major factors limiting the replication efficiency of oncolytic viruses are the antiviral defense pathways activated by tumor cells. In this study, we have designed and validated a universal expression cassette for artificial microRNAs that can now be adapted to suppress genes of interest, including potential resistance factors. Transcripts are encoded as a primary microRNA for processing via the predominantly nuclear RNase III Drosha. We have engineered an oncolytic measles virus encoding this universal expression cassette for artificial microRNAs. Virally encoded microRNA was expressed in the range of endogenous microRNA transcripts and successfully mediated target protein suppression. However, absolute expression levels of mature microRNAs were limited when delivered by an oncolytic measles virus. We demonstrate that measles virus, in contrast to other cytosolic viruses, does not induce translocation of Drosha from the nucleus into the cytoplasm, potentially resulting in a limited processing efficiency of virus-derived, cytosolically delivered artificial microRNAs. To our knowledge, this is the first report demonstrating functional expression of microRNA from oncolytic measles viruses potentially enabling future targeted knockdown, for instance of antiviral factors specifically in tumor cells.
Topics: Humans; Measles virus; MicroRNAs; RNA Interference; Oncolytic Viruses; Measles; Antiviral Agents
PubMed: 36851522
DOI: 10.3390/v15020308 -
The Journal of Cell Biology Jul 2008MicroRNAs (miRNAs) are noncoding RNAs with important roles in regulating gene expression. In studying the earliest nuclear steps of miRNA biogenesis, we observe that...
MicroRNAs (miRNAs) are noncoding RNAs with important roles in regulating gene expression. In studying the earliest nuclear steps of miRNA biogenesis, we observe that primary miRNA (pri-miRNA) transcripts retained at transcription sites due to the deletion of 3'-end processing signals are converted more efficiently into precursor miRNAs (pre-miRNAs) than pri-miRNAs that are cleaved, polyadenylated, and released. Flanking exons, which also increase retention at transcription sites, likewise contribute to increased levels of intronic pri-miRNAs. Consistently, efficiently processed endogenous pri-miRNAs are enriched in chromatin-associated nuclear fractions. In contrast, pri-miRNAs that accumulate to high nuclear levels after cleavage and polyadenylation because of the presence of a viral RNA element (the ENE of the Kaposi's sarcoma-associated herpes virus polyadenylated nuclear RNA) are not efficiently processed to precursor or mature miRNAs. Exogenous pri-miRNAs unexpectedly localize to nuclear foci containing splicing factor SC35; yet these foci are unlikely to represent sites of miRNA transcription or processing. Together, our results suggest that pri-miRNA processing is enhanced by coupling to transcription.
Topics: Chromatin; Exons; HeLa Cells; Humans; Introns; MicroRNAs; Nuclear Proteins; Polyadenylation; RNA Polymerase III; RNA Processing, Post-Transcriptional; RNA Transport; RNA, Messenger; RNA, Viral; Regulatory Sequences, Nucleic Acid; Ribonucleoproteins; Sequence Deletion; Serine-Arginine Splicing Factors; Subcellular Fractions; Transcription, Genetic; mRNA Cleavage and Polyadenylation Factors
PubMed: 18625843
DOI: 10.1083/jcb.200803111 -
Mutation Research. Reviews in Mutation... 2015Multiple sclerosis (MS) is a neurological immune-driven disease of unknown etiology. A genetic contribution to MS susceptibility is well established and more than 100... (Review)
Review
Multiple sclerosis (MS) is a neurological immune-driven disease of unknown etiology. A genetic contribution to MS susceptibility is well established and more than 100 associated genomic regions have been identified. However, there is little understanding of how individual genetic variants are involved in the pathogenesis of MS. Single nucleotide polymorphisms (SNPs) within the first intron of CD58 have been independently confirmed to be related to the risk of MS. We here provide evidence that these SNPs may implicate an altered processing of an intronic microRNA. This microRNA, hsa-mir-548ac, belongs to a huge primate-specific family of microRNAs that evolved from a mariner-derived transposable element (Made1). Sequencing data of the 1000 Genomes project revealed a SNP, rs1414273, at the base of the microRNA stem-loop to be in strong linkage disequilibrium with the MS-associated haplotype. This SNP is suspected to affect the recognition of the primary microRNA hairpin by Drosha and its cofactor DGCR8. Future studies on microRNA-548ac and its genetic variant may yield deeper insights into the mechanisms underlying MS. Experimental challenges and open questions are discussed.
Topics: Animals; CD58 Antigens; DNA Transposable Elements; Evolution, Molecular; Genetic Association Studies; Genetic Predisposition to Disease; Genetic Variation; Humans; Introns; Linkage Disequilibrium; MicroRNAs; Multiple Sclerosis; Polymorphism, Single Nucleotide; Primates; RNA-Binding Proteins; Ribonuclease III
PubMed: 25795118
DOI: 10.1016/j.mrrev.2014.10.002 -
Pediatric Research Jan 2024Increased maternal interleukin (IL)-17A and activated microglia are pivotal factors contributing to the pathological phenotypes of maternal immune activation (MIA),...
BACKGROUND
Increased maternal interleukin (IL)-17A and activated microglia are pivotal factors contributing to the pathological phenotypes of maternal immune activation (MIA), developing neurodevelopmental disorders in offspring. This study aimed to determine whether IL-17A affects the microglial microRNA (miRNA) profiles.
METHODS
The miRNA expression profiles of primary cultured microglia stimulated with recombinant IL-17A were examined comprehensively using miRNA sequencing and validated through qRT-PCR. The expressions of miRNAs target genes identified using bioinformatics, were investigated in microglia transfected with mimic miRNA. The target gene's expression was also examined in the fetal brains of the MIA mouse model induced by maternal lipopolysaccharide (LPS) administration.
RESULTS
Primary cultured microglia expressed the IL-17A receptor and increased proinflammatory cytokines and nitric oxide synthase 2 upon treatment with IL-17A. Among the three miRNAs with |logFC | >1, only mmu-miR-206-3p expression was significantly up-regulated by IL-17A. Transfection with the mmu-miR-206-3p mimic resulted in a significant decrease in the expression of Hdac4 and Igf1, target genes of mmu-miR-206-3p. Hdac4 expression also significantly decreased in the LPS-induced MIA model.
CONCLUSIONS
IL-17A affected microglial miRNA profiles with upregulated mmu-miR-206-3p. These findings suggest that targeting the IL-17A/mmu-miR-206-3p pathway may be a new strategy for predicting MIA-related neurodevelopmental deficits and providing preventive interventions.
IMPACT
Despite the growing evidence of interleukin (IL)-17A and microglia in the pathology of maternal immune activation (MIA), the downstream of IL-17A in microglia is not fully known. IL-17A altered microRNA profiles and upregulated the mmu-miR-206-3p expression in microglia. The mmu-miR-206-3p reduced autism spectrum disorder (ASD) related gene expressions, Hdac4 and Igf1. The Hdac4 expression was also reduced in the brain of MIA offspring. The hsa-miR-206 sequence is consistent with that of mmu-miR-206-3p. This study may provide clues to pathological mechanisms leading to predictions and interventions for ASD children born to mothers with IL-17A-related disorders.
Topics: Mice; Animals; Child; Humans; Microglia; Interleukin-17; Autism Spectrum Disorder; Lipopolysaccharides; MicroRNAs
PubMed: 37758861
DOI: 10.1038/s41390-023-02825-6 -
The Journal of Biological Chemistry Apr 2022High plasma lipid levels have been demonstrated to increase cardiovascular disease risk. Despite advances in treatments to decrease plasma lipids, additional...
High plasma lipid levels have been demonstrated to increase cardiovascular disease risk. Despite advances in treatments to decrease plasma lipids, additional therapeutics are still needed because many people are intolerant or nonresponsive to these therapies. We previously showed that increasing cellular levels of microRNA-30c (miR-30c) using viral vectors or liposomes reduces plasma lipids and atherosclerosis. In this study, we aimed to synthesize potent miR-30c analogs that can be delivered to hepatoma cells without the aid of viral vectors and lipid emulsions. We hypothesized that modification of the passenger strand of miR-30c would increase the stability of miR-30c and augment its delivery to liver cells. Here, we report the successful synthesis of a series of miR-30c analogs by using different chemically modified nucleosides. In these analogs, we left the active sense strand untouched so that its biological activity remained unaltered, and we modified the passenger strand of miR-30c to enhance the stability and uptake of miR-30c by hepatoma cells through phosphorothiorate linkages and the addition of GalNAc. We show that these analogs significantly reduced apolipoprotein B secretion in Huh-7 human hepatoma cells and human primary hepatocytes without affecting apolipoprotein A1 secretion and cellular lipid levels. Our results provide a proof of concept that the passenger strand of miR-30c can be modified to increase its stability and delivery to cells while retaining the potency of the sense strand. We anticipate these miR-30c analogs will be useful in the development of more efficacious analogs for the treatment of hyperlipidemias and cardiovascular diseases.
Topics: Apolipoproteins B; Carcinoma, Hepatocellular; Cells, Cultured; Hepatocytes; Humans; Liver Neoplasms; MicroRNAs
PubMed: 35278429
DOI: 10.1016/j.jbc.2022.101813 -
Mutagenesis Sep 2016The well-defined battery of in vitro systems applied within chemical cancer risk assessment is often characterised by a high false-positive rate, thus repeatedly failing...
The well-defined battery of in vitro systems applied within chemical cancer risk assessment is often characterised by a high false-positive rate, thus repeatedly failing to correctly predict the in vivo genotoxic and carcinogenic properties of test compounds. Toxicogenomics, i.e. mRNA-profiling, has been proven successful in improving the prediction of genotoxicity in vivo and the understanding of underlying mechanisms. Recently, microRNAs have been discovered as post-transcriptional regulators of mRNAs. It is thus hypothesised that using microRNA response-patterns may further improve current prediction methods. This study aimed at predicting genotoxicity and non-genotoxic carcinogenicity in vivo, by comparing microRNA- and mRNA-based profiles, using a frequently applied in vitro liver model and exposing this to a range of well-chosen prototypical carcinogens. Primary mouse hepatocytes (PMH) were treated for 24 and 48h with 21 chemical compounds [genotoxins (GTX) vs. non-genotoxins (NGTX) and non-genotoxic carcinogens (NGTX-C) versus non-carcinogens (NC)]. MicroRNA and mRNA expression changes were analysed by means of Exiqon and Affymetrix microarray-platforms, respectively. Classification was performed by using Prediction Analysis for Microarrays (PAM). Compounds were randomly assigned to training and validation sets (repeated 10 times). Before prediction analysis, pre-selection of microRNAs and mRNAs was performed by using a leave-one-out t-test. No microRNAs could be identified that accurately predicted genotoxicity or non-genotoxic carcinogenicity in vivo. However, mRNAs could be detected which appeared reliable in predicting genotoxicity in vivo after 24h (7 genes) and 48h (2 genes) of exposure (accuracy: 90% and 93%, sensitivity: 65% and 75%, specificity: 100% and 100%). Tributylinoxide and para-Cresidine were misclassified. Also, mRNAs were identified capable of classifying NGTX-C after 24h (5 genes) as well as after 48h (3 genes) of treatment (accuracy: 78% and 88%, sensitivity: 83% and 83%, specificity: 75% and 93%). Wy-14,643, phenobarbital and ampicillin trihydrate were misclassified. We conclude that genotoxicity and non-genotoxic carcinogenicity probably cannot be accurately predicted based on microRNA profiles. Overall, transcript-based prediction analyses appeared to clearly outperform microRNA-based analyses.
Topics: Animals; Carcinogens; Hepatocytes; Male; Mice; MicroRNAs; Mutagenicity Tests; Proteins; RNA, Messenger; Sensitivity and Specificity; Toxicogenetics; Transcriptome
PubMed: 27338304
DOI: 10.1093/mutage/gew027 -
Biological Psychiatry Jan 2015
Topics: Animals; Brain; Embryo, Mammalian; Embryonic Development; Fetal Diseases; Mice, Inbred C57BL; Mice, Knockout; MicroRNAs; Polymerase Chain Reaction
PubMed: 25016316
DOI: 10.1016/j.biopsych.2014.05.022 -
Cell Jan 2016MicroRNA maturation is initiated by RNase III DROSHA that cleaves the stem loop of primary microRNA. DROSHA functions together with its cofactor DGCR8 in a...
MicroRNA maturation is initiated by RNase III DROSHA that cleaves the stem loop of primary microRNA. DROSHA functions together with its cofactor DGCR8 in a heterotrimeric complex known as Microprocessor. Here, we report the X-ray structure of DROSHA in complex with the C-terminal helix of DGCR8. We find that DROSHA contains two DGCR8-binding sites, one on each RNase III domain (RIIID), which mediate the assembly of Microprocessor. The overall structure of DROSHA is surprisingly similar to that of Dicer despite no sequence homology apart from the C-terminal part, suggesting that DROSHA may have evolved from a Dicer homolog. DROSHA exhibits unique features, including non-canonical zinc-finger motifs, a long insertion in the first RIIID, and the kinked link between Connector helix and RIIID, which explains the 11-bp-measuring "ruler" activity of DROSHA. Our study implicates the evolutionary origin of DROSHA and elucidates the molecular basis of Microprocessor assembly and primary microRNA processing.
Topics: Amino Acid Sequence; Crystallography, X-Ray; DEAD-box RNA Helicases; Evolution, Molecular; Humans; MicroRNAs; Models, Chemical; Models, Molecular; Molecular Sequence Data; Nucleic Acid Conformation; Protein Folding; Protein Structure, Tertiary; RNA Processing, Post-Transcriptional; RNA-Binding Proteins; Ribonuclease III; Sequence Alignment; Structural Homology, Protein
PubMed: 26748718
DOI: 10.1016/j.cell.2015.12.019