RESUMEN
Many microRNAs regulate gene expression via atypical mechanisms, which are difficult to discern using native cross-linking methods. To ascertain the scope of non-canonical miRNA targeting, methods are needed that identify all targets of a given miRNA. We designed a new class of miR-CLIP probe, whereby psoralen is conjugated to the 3p arm of a pre-microRNA to capture targetomes of miR-124 and miR-132 in HEK293T cells. Processing of pre-miR-124 yields miR-124 and a 5'-extended isoform, iso-miR-124. Using miR-CLIP, we identified overlapping targetomes from both isoforms. From a set of 16 targets, 13 were differently inhibited at mRNA/protein levels by the isoforms. Moreover, delivery of pre-miR-124 into cells repressed these targets more strongly than individual treatments with miR-124 and iso-miR-124, suggesting that isomirs from one pre-miRNA may function synergistically. By mining the miR-CLIP targetome, we identified nine G-bulged target-sites that are regulated at the protein level by miR-124 but not isomiR-124. Using structural data, we propose a model involving AGO2 helix-7 that suggests why only miR-124 can engage these sites. In summary, access to the miR-124 targetome via miR-CLIP revealed for the first time how heterogeneous processing of miRNAs combined with non-canonical targeting mechanisms expand the regulatory range of a miRNA.
Asunto(s)
Proteínas Argonautas/metabolismo , Regulación de la Expresión Génica , MicroARNs/genética , Modelos Genéticos , Regiones no Traducidas 3'/genética , Secuencias de Aminoácidos , Proteínas Argonautas/química , Secuencia de Bases , Sitios de Unión , Biotina , Reactivos de Enlaces Cruzados/farmacología , ADN Complementario/genética , Proteínas de Unión al GTP/genética , Células HEK293 , Humanos , Inmunoprecipitación , MicroARNs/antagonistas & inhibidores , Proteínas Nucleares/genética , Conformación de Ácido Nucleico , Fotoquímica , Análisis de Secuencia de ADN , Estreptavidina , Trioxsaleno/efectos de la radiaciónRESUMEN
The oligonucleotide therapeutics field has blossomed in recent years, with thirteen approved drugs today and the promise of accelerated growth in coming years. Much of the progress in this field is due to advances in the medicinal chemistry of oligonucleotides,combined with a judicious choice of molecular targets and disease areas. In this perspective, we describe the growth of this new class of drugs highlighting selected milestones in oligonucleotide medicinal chemistry.
RESUMEN
It is well-accepted that gene expression is heavily influenced by RNA structure. For instance, stem-loops and G-quadruplexes (rG4s) are dynamic motifs in mRNAs that influence gene expression. Adenosine-to-inosine (A-to-I) editing is a common chemical modification of RNA which introduces a nucleobase that is iso-structural with guanine, thereby changing RNA base-pairing properties. Here, we provide biophysical, chemical, and biological evidence that A-to-I exchange can activate latent rG4s by filling incomplete G-quartets with inosine. We demonstrate the formation of inosine-containing rG4s (GI-quadruplexes) in vitro and verify their activity in cells. GI-quadruplexes adopt parallel topologies, stabilized by potassium ions. They exhibit moderately reduced thermal stability compared to conventional G-quadruplexes. To study inosine-induced structural changes in a naturally occurring RNA, we use a synthetic approach that enables site-specific inosine incorporation in long RNAs. In summary, RNA GI-quadruplexes are a previously unrecognized structural motif that may contribute to the regulation of gene expression in vivo.
Asunto(s)
G-Cuádruplex , Inosina/química , ARN/química , Emparejamiento Base , Regulación de la Expresión Génica/efectos de los fármacos , Células HEK293 , Humanos , Conformación de Ácido NucleicoRESUMEN
MicroRNAs constitute a class of endogenous, non-coding RNAs that influence various processes within the cell. By base-pairing to partially-complementary sites located in the 3' untranslated region of target messenger RNAs, microRNAs participate in post-transcriptional regulation of the majority of human protein-coding genes. Their dysregulation has been related to many pathological processes and diseases. Thus, an in-depth understanding of the microRNA mechanisms of action is crucial. Here, we present a new concept of probe design to achieve an efficient and sequence-independent miRNA-mRNA cross-linking. The new strategy is based on the utilization of a controlled mixture of probes for a chosen miRNA, in which a trioxsalen moiety is introduced at the N4 -position of a selected cytidine through short oligoethylene glycol-based linkers. In vitro photo-cross-linking experiments with mini-libraries of probes for microRNAs of interest showed variable cross-linking efficiencies, demonstrating a general applicability of the presented approach.
Asunto(s)
MicroARNs , Regiones no Traducidas 3' , Emparejamiento Base , Regulación de la Expresión Génica , Humanos , MicroARNs/genética , ARN Mensajero/genéticaRESUMEN
Lin28 RNA-binding proteins are stem-cell factors that play key roles in development. Lin28 suppresses the biogenesis of let-7 microRNAs and regulates mRNA translation. Notably, let-7 inhibits Lin28, establishing a double-negative feedback loop. The Lin28/let-7 axis resides at the interface of metabolic reprogramming and oncogenesis and is therefore a potential target for several diseases. In this study, we use compound-C1632, a drug-like Lin28 inhibitor, and show that the Lin28/let-7 axis regulates the balance between ketogenesis and lipogenesis in liver cells. Hence, Lin28 inhibition activates synthesis and secretion of ketone bodies whilst suppressing lipogenesis. This occurs at least partly via let-7-mediated inhibition of nuclear receptor co-repressor 1, which releases ketogenesis gene expression mediated by peroxisome proliferator-activated receptor-alpha. In this way, small-molecule Lin28 inhibition protects against lipid accumulation in multiple cellular and male mouse models of hepatic steatosis. Overall, this study highlights Lin28 inhibitors as candidates for the treatment of hepatic disorders of abnormal lipid deposition.
Asunto(s)
MicroARNs , Enfermedad del Hígado Graso no Alcohólico , Animales , Ratones , Masculino , MicroARNs/genética , MicroARNs/metabolismo , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Enfermedad del Hígado Graso no Alcohólico/genética , Homeostasis , LípidosRESUMEN
Identifying the targetome of a microRNA is key for understanding its functions. Cross-linking and immunoprecipitation (CLIP) methods capture native miRNA-mRNA interactions in cells. Some of these methods yield small amounts of chimeric miRNA-mRNA sequences via ligation of 5'-phosphorylated RNAs produced during the protocol. Here, we introduce chemically synthesized microRNAs (miRNAs) bearing 2'-, 3'-cyclic phosphate groups, as part of a new CLIP method that does not require 5'-phosphorylation for ligation. We show in a system that models miRNAs bound to their targets, that addition of recombinant bacterial ligase RtcB increases ligation efficiency, and that the transformation proceeds via a 3'-phosphate intermediate. By optimizing the chemistry underlying ligation, we provide the basis for an improved method to identify miRNA targetomes.
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MicroARNs/química , MicroARNs/metabolismo , ARN Mensajero/química , ARN Mensajero/metabolismo , Proteínas Bacterianas/metabolismo , Sitios de Unión , Reactivos de Enlaces Cruzados/química , Células HEK293 , Células HeLa , Humanos , Inmunoprecipitación , Ligasas/metabolismo , Fosforilación , Proteínas Recombinantes/metabolismoRESUMEN
Following organ transplantation, it is essential that immune tolerance is induced in the graft recipient to reduce the risk of rejection and avoid complications associated with the long-term use of immunosuppressive drugs. Immature dendritic cells (DCs) are considered to promote transplant tolerance and may minimize the risk of graft rejection. The aim of the study was to evaluate the effects of immunosuppressive agents: rapamycin (Rapa) and cyclosporine A (CsA) on generation of human tolerogenic DCs (tolDCs) and also to evaluate the ability of these cells to induce mechanisms of immune tolerance. tolDCs were generated in the environment of Rapa or CsA. Next, we evaluated the effects of these agents on surface phenotypes (CD11c, MHC II, CD40, CD80, CD83, CD86, CCR7, TLR2, TLR4), cytokine production (IL-4, IL-6, IL-10, IL-12p70, TGF-ß), phagocytic capacity and resistant to lipopolysaccharide activation of these DCs. Moreover, we assessed ability of such tolDCs to induce T cell activation and apoptosis, Treg differentiation and production of Th1- and Th2-characteristic cytokine profile. Data obtained in this study demonstrate that rapamycin is effective at generating maturation-resistant tolDCs, however, does not change the ability of these cells to induce mechanisms of immune tolerance. In contrast, CsA affects the ability of these cells to induce mechanisms of immune tolerance, but is not efficient at generating maturation-resistant tolDCs.
Asunto(s)
Ciclosporina , Sirolimus , Células Dendríticas , Humanos , Tolerancia Inmunológica , InmunosupresoresRESUMEN
Objectives: Macrophages are conventionally classified as pro-inflammatory (M1) and anti-inflammatory (M2) functional types. There is evidence for a predominance of macrophages with an inflammatory phenotype (M1) in the rheumatoid arthritis (RA) synovium. MicroRNAs (miRs) play a pivotal role in regulating the inflammatory response in innate immune cells and are found at dysregulated levels in RA patients. Here we explored miRs that tune the inflammatory function of M2-macrophages. Methods: Expression profiles of miR-221-3p and miR-155-5p were analyzed in clinical samples from RA, other inflammatory arthritis (OIA), osteoarthritis (OA), and healthy donors (HD) by qPCR. In vitro generated macrophages were transfected with miR-mimics and inhibitors. Transcriptome profiling through RNA-sequencing was performed on M2-macrophages overexpressing miR-221-3p mimic with or without LPS treatment. Secretion of IL-6, IL-10, IL-12, IL-8, and CXCL13 was measured in M1- and M2-macrophages upon TLR2/TLR3/TLR4-stimulation using ELISA. Inflammatory pathways including NF-κB, IRF3, MAPKs, and JAK3/STAT3 were evaluated by immunoblotting. Direct target interaction of miR-221-3p and predicted target sites in 3'UTR of JAK3 were examined by luciferase reporter gene assay. Results: miR-221-3p in synovial tissue and fluid was increased in RA vs. OA or OIA. Endogenous expression levels of miR-221-3p and miR-155-5p were higher in M1- than M2-macrophages derived from RA patients or HD. TLR4-stimulation of M1- and M2-macrophages resulted in downregulation of miR-221-3p, but upregulation of miR-155-5p. M2-macrophages transfected with miR-221-3p mimics secreted less IL-10 and CXCL13 but more IL-6 and IL-8, exhibited downregulation of JAK3 protein and decreased pSTAT3 activation. JAK3 was identified as new direct target of miR-221-3p in macrophages. Co-transfection of miR-221-3p/miR-155-5p mimics in M2-macrophages increased M1-specific IL-12 secretion. Conclusions: miR-221-3p acts as a regulator of TLR4-induced inflammatory M2-macrophage function by directly targeting JAK3. Dysregulated miR-221-3p expression, as seen in synovium of RA patients, leads to a diminished anti-inflammatory response and drives M2-macrophages to exhibit a M1-cytokine profile.
Asunto(s)
Regulación de la Expresión Génica , Janus Quinasa 3/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , MicroARNs/genética , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Regiones no Traducidas 3' , Biomarcadores , Plasticidad de la Célula , Citocinas/metabolismo , Perfilación de la Expresión Génica , Humanos , Mediadores de Inflamación/metabolismo , Interferencia de ARN , Receptor Toll-Like 4/genéticaRESUMEN
The DIR2s RNA aptamer, a second-generation, in-vitro selected binder to dimethylindole red (DIR), activates the fluorescence of cyanine dyes, DIR and oxazole thiazole blue (OTB), allowing detection of two well-resolved emission colors. Using Fab BL3-6 and its cognate hairpin as a crystallization module, we solved the crystal structures of both the apo and OTB-SO3 bound forms of DIR2s at 2.0 Å and 1.8 Å resolution, respectively. DIR2s adopts a compact, tuning fork-like architecture comprised of a helix and two short stem-loops oriented in parallel to create the ligand binding site through tertiary interactions. The OTB-SO3 fluorophore binds in a planar conformation to a claw-like structure formed by a purine base-triple, which provides a stacking platform for OTB-SO3, and an unpaired nucleotide, which partially caps the binding site from the top. The absence of a G-quartet or base tetrad makes the DIR2s aptamer unique among fluorogenic RNAs with known 3D structure.