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1.
Nucleic Acids Res ; 49(5): 2522-2536, 2021 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-33561291

RESUMO

Simultaneous dysregulation of multiple microRNAs (miRs) affects various pathological pathways related to cardiac failure. In addition to being potential cardiac disease-specific markers, miR-23b/27b/24-1 were reported to be responsible for conferring cardiac pathophysiological processes. In this study, we identified a conserved guanine-rich RNA motif within the miR-23b/27b/24-1 cluster that can form an RNA G-quadruplex (rG4) in vitro and in cells. Disruption of this intragenic rG4 significantly increased the production of all three miRs. Conversely, a G4-binding ligand tetrandrine (TET) stabilized the rG4 and suppressed miRs production in human and rodent cardiomyocytes. Our further study showed that the rG4 prevented Drosha-DGCR8 binding and processing of the pri-miR, suppressing the biogenesis of all three miRs. Moreover, CRISPR/Cas9-mediated G4 deletion in the rat genome aberrantly elevated all three miRs in the heart in vivo, leading to cardiac contractile dysfunction. Importantly, loss of the G4 resulted in reduced targets for the aforementioned miRs critical for normal heart function and defects in the L-type Ca2+ channel-ryanodine receptor (LCC-RyR) coupling in cardiomyocytes. Our results reveal a novel mechanism for G4-dependent regulation of miR biogenesis, which is essential for maintaining normal heart function.


Assuntos
Quadruplex G , MicroRNAs/química , MicroRNAs/metabolismo , Contração Miocárdica/genética , Miócitos Cardíacos/metabolismo , Animais , Benzilisoquinolinas/farmacologia , Sistemas CRISPR-Cas , Células Cultivadas , Quadruplex G/efeitos dos fármacos , Regulação da Expressão Gênica , Miocárdio/metabolismo , Miócitos Cardíacos/fisiologia , Processamento Pós-Transcricional do RNA , Proteínas de Ligação a RNA/metabolismo , Ratos , Ratos Sprague-Dawley , Ribonuclease III/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo
2.
Anal Chem ; 93(4): 2226-2234, 2021 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-33417427

RESUMO

Real-time imaging of multiple low-abundance microRNAs (miRNAs) simultaneously in living cells with high sensitivity is of vital importance for accurate cancer clinical diagnosis and prognosis studies. Maintaining stability of nanoprobes resistant to enzyme degradation and enabling effective signal amplification is highly needed for in vivo imaging studies. Herein, a rationally designed one-pot assembled multicolor tetrahedral DNA frameworks (TDFs) by encoding multicomponent nucleic acid enzymes (MNAzymes) was developed for signal-amplified multiple miRNAs imaging in living cells with high sensitivity and selectivity. TDFs could enter cells via self-delivery with good biocompatibility and stability. Two kinds of MNAzymes specific for miRNA-21 and miRNA-155 with fluorescein labeling were encoded in the structure of TDFs respectively through one-step thermal annealing. In the intracellular environment, the TDFs could be specifically bound with its specific miRNA target and form an active DNAzyme structure. The cleavage of the active site would trigger the release of target miRNA and circular fluorescence signal amplification, which enabled accurate diagnosis on miRNA identifications of different cell lines with high sensitivity. Meanwhile, with the specific AS1411 aptamer targeting for nucleolin overexpressed on the surface of the carcinoma cells, this well-designed TDFs nanoprobe exhibited good discrimination between cancer cells and normal cells. The strategy provides an efficient tool for understanding the biological function of miRNAs in cancer pathogenesis and therapeutic applications.


Assuntos
DNA/química , MicroRNAs/química , Imagem Molecular/métodos , Linhagem Celular Tumoral , Sobrevivência Celular , Humanos , Espaço Intracelular/metabolismo , Microscopia de Força Atômica , Sondas Moleculares/química , Nanotecnologia/métodos , Conformação de Ácido Nucleico
3.
Anal Chem ; 93(4): 2038-2044, 2021 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-33411517

RESUMO

The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) (CRISPR/Cas) system innovates a next-generation biosensor due to its high-fidelity, programmability, and efficient signal amplification ability. Developing a CRISPR/Cas-based visual detection system could contribute to point-of-care biomarker diagnosis. Existing CRISPR/Cas9-mediated visual detection methods are limited by the inherent properties of Cas9. Herein, we explored the trans-cleavage ability of Cas13a on ribonucleotide-bearing DNA oligo, eliminated the unavailability of the trans-cleavage substrate for subsequent polymerization reaction, and developed a homogeneous CRISPR/Cas13a-based visual detection system (termed vCas) for specific and sensitive detection of miRNA. The results indicated that vCas can provide a detection limit of 1 fM for miR-10b with single-base specificity and can be used to analyze miRNA in serum and cell extracts. Conclusively, vCas holds a great application prospective for clinical molecular diagnosis.


Assuntos
Sistemas CRISPR-Cas , DNA Catalítico/metabolismo , MicroRNAs/química , Técnicas Biossensoriais , Linhagem Celular , DNA Catalítico/genética , Humanos , Conformação Proteica
4.
Anal Chem ; 93(4): 2519-2526, 2021 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-33404216

RESUMO

MicroRNAs (miRNAs) in exosomes can be transferred from parental cells to recipient cells by trafficking exosomes, and they are effective in regulating the gene expression of the recipient cells. Therefore, exosomal miRNAs play a vital role in cancer biology and could be potential biomarkers for cancer diagnosis and therapeutic responses. However, accurate detection of exosomal miRNAs is still challenging due to the low abundance of any given miRNA in exosomes. Herein, a surface-enhanced Raman scattering (SERS)-based sensor was developed for the quantitative determination of let-7a miRNAs in MCF-7 cell-derived exosomes (MCF-7 exosomes) using a close-packed and ordered Au octahedral array as a sensing platform. Au octahedra in the array uniformly stand on their triangular face. This kind of orientation produces "hot surfaces" rather than "hot spots" and greatly improves the detection sensitivity and uniformity. Let-7a detection with single-base specificity was thus achieved from the SERS intensity change induced by the structural switch of the probing DNA from a hairpin to a duplex in the presence of the target. The sensor showed a broad linear range (10 aM to 10 nM) and a low detection limit (5.3 aM) without using any signal amplification strategy. Moreover, this sensor could accurately detect target let-7a in MCF-7 exosomes and further value the impact of drug treatment on exosomal let-7a expression, indicating promising applications of the developed sensor for cancer diagnostics and therapy.


Assuntos
Ouro , MicroRNAs/química , Análise Espectral Raman/instrumentação , Análise Espectral Raman/métodos , Técnicas Biossensoriais , Exossomos , Humanos , Células MCF-7 , Sensibilidade e Especificidade
5.
Anal Chem ; 93(4): 2534-2540, 2021 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-33461295

RESUMO

MicroRNAs (miRNAs) play essential roles in regulating gene expression and cell fate. However, it remains a great challenge to image miRNAs with high accuracy in living cells. Here, we report a novel genetically encoded dual-color light-up RNA sensor for ratiometric imaging of miRNAs using Mango as an internal reference and SRB2 as the sensor module. This genetically encoded sensor is designed by expressing a splittable fusion of the internal reference and sensor module under a single promoter. This design strategy allows synchronous expression of the two modules with negligible interference. Live cell imaging studies reveal that the genetically encoded ratiometric RNA sensor responds specifically to mir-224. Moreover, the sensor-to-Mango fluorescence ratios are linearly correlated with the concentrations of mir-224, confirming their capability of determining mir-224 concentrations in living cells. Our genetically encoded light-up RNA sensor also enables ratiometric imaging of mir-224 in different cell lines. This strategy could provide a versatile approach for ratiometric imaging of intracellular RNAs, affording powerful tools for interrogating RNA functions and abundance in living cells.


Assuntos
Proteínas Luminescentes/genética , MicroRNAs/química , Imagem Óptica/métodos , RNA/química , Técnicas Biossensoriais , Linhagem Celular , Engenharia Genética/métodos , Humanos , Imagem Molecular/métodos
6.
Viruses ; 13(1)2021 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-33467206

RESUMO

Our recent study identified seven key microRNAs (miR-8066, 5197, 3611, 3934-3p, 1307-3p, 3691-3p, 1468-5p) similar between SARS-CoV-2 and the human genome, pointing at miR-related mechanisms in viral entry and the regulatory effects on host immunity. To identify the putative roles of these miRs in zoonosis, we assessed their conservation, compared with humans, in some key wild and domestic animal carriers of zoonotic viruses, including bat, pangolin, pig, cow, rat, and chicken. Out of the seven miRs under study, miR-3611 was the most strongly conserved across all species; miR-5197 was the most conserved in pangolin, pig, cow, bat, and rat; miR-1307 was most strongly conserved in pangolin, pig, cow, bat, and human; miR-3691-3p in pangolin, cow, and human; miR-3934-3p in pig and cow, followed by pangolin and bat; miR-1468 was most conserved in pangolin, pig, and bat; while miR-8066 was most conserved in pangolin and pig. In humans, miR-3611 and miR-1307 were most conserved, while miR-8066, miR-5197, miR-3334-3p and miR-1468 were least conserved, compared with pangolin, pig, cow, and bat. Furthermore, we identified that changes in the miR-5197 nucleotides between pangolin and human can generate three new miRs, with differing tissue distribution in the brain, lung, intestines, lymph nodes, and muscle, and with different downstream regulatory effects on KEGG pathways. This may be of considerable importance as miR-5197 is localized in the spike protein transcript area of the SARS-CoV-2 genome. Our findings may indicate roles for these miRs in viral-host co-evolution in zoonotic hosts, particularly highlighting pangolin, bat, cow, and pig as putative zoonotic carriers, while highlighting the miRs' roles in KEGG pathways linked to viral pathogenicity and host responses in humans. This in silico study paves the way for investigations into the roles of miRs in zoonotic disease.


Assuntos
Coevolução Biológica , MicroRNAs/genética , /genética , Animais , /virologia , Galinhas , Redes Reguladoras de Genes , Genoma/genética , Especificidade de Hospedeiro , Humanos , Mamíferos , MicroRNAs/química , MicroRNAs/metabolismo , /fisiologia , Alinhamento de Sequência , Distribuição Tecidual , Zoonoses/transmissão , Zoonoses/virologia
7.
ACS Appl Mater Interfaces ; 13(5): 6034-6042, 2021 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-33499584

RESUMO

MicroRNA (miRNA) represents a promising class of therapeutic nucleic acid drugs, while delivery challenges remain that impede the advancement of miRNA therapy, largely because of in vivo instability and low delivery efficiency. Herein, we discover the dual roles of metal-organic framework (MOF) nanoparticles (ZIF-8) as nanocarriers for miRNA delivery and adjuvants for chemodynamic therapy. The miR-34a-m@ZIF-8 complex demonstrated efficient cellular uptake and lysosomal stimuli-responsive miRNA release. Zn2+ triggered the generation of reactive oxygen species, which consequently induced apoptosis of tumor cells. Released miR-34a-m led to a remarkable decrease in expression of Bcl-2 at both mRNA and protein levels and enhanced cancer cell apoptosis. In vivo experiments showed high efficacy of using miR-34a-m@ZIF-8 to suppress tumor growth via synergistic gene/chemodynamic therapy in a mouse model of triple-negative breast cancer. Our work demonstrates MOFs as a promising nanoplatform for efficient synergetic gene/chemodynamic therapy.


Assuntos
Adjuvantes Farmacêuticos/farmacologia , Antibióticos Antineoplásicos/farmacologia , Doxorrubicina/farmacologia , Sistemas de Liberação de Medicamentos , Estruturas Metalorgânicas/química , MicroRNAs/farmacologia , Nanopartículas/química , Adjuvantes Farmacêuticos/química , Animais , Antibióticos Antineoplásicos/química , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/química , Portadores de Fármacos/química , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Neoplasias Mamárias Experimentais/diagnóstico por imagem , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/metabolismo , Camundongos , MicroRNAs/química , Tamanho da Partícula , Espécies Reativas de Oxigênio/metabolismo , Propriedades de Superfície , Zeolitas/química , Zeolitas/farmacologia
8.
Methods Mol Biol ; 2170: 143-154, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32797457

RESUMO

MicroRNAs (miRNAs) play important roles in development in plants, and some miRNAs show developmentally regulated organ- and tissue-specific expression patterns. Therefore, in situ detection of mature miRNAs is important for understanding the functions for both miRNAs and their targets. The construction of promoter-reporter fusions and examination of their in planta expression has been widely used and the results obtained thus far are rather informative; however, in some cases, the length of promoter that contains entire regulatory elements is difficult to determine. In addition, traditional in situ hybridization with the antisense RNA fragment as the probe usually fails to detect miRNAs, because the mature miRNAs are too short (~21-nucleotides) to exhibit stable hybridization signals. In recent years, the Locked nucleic acid (LNA) modified DNA probe has been successfully used in animals and plants to detect small RNAs. Here, we describe a modified protocol using LNA-modified DNA probes to detect mature miRNAs in plant tissues, including the design of LNA probes and detailed steps for the in situ hybridization experiment, using Arabidopsis miR165 as an example.


Assuntos
Sondas de DNA/química , MicroRNAs/análise , MicroRNAs/química , Oligonucleotídeos/química , RNA de Plantas/análise , RNA de Plantas/química , Hibridização In Situ
9.
Virus Res ; 294: 198275, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33359190

RESUMO

Tmprss2 is an emerging molecular target which guides cellular infections of SARS-CoV-2, has been earmarked for interventions against the viral pathologies. The study aims to computationally screen and identifies potential miRNAs, following in vitro experimental validation of miRNA-mediated suppression of Tmprss2 for early prevention of COVID-19. Pool of 163 miRNAs, scrutinized for Tmprss2 binding with three miRNA prediction algorithms, ensued 11 common miRNAs. Further, computational negative energies for association, corroborated miRNA-Tmprss2 interactions, whereas three miRNAs (hsa-miR-214, hsa-miR-98 and hsa-miR-32) based on probability scores ≥0.8 and accessibility to Tmprss2 target have been selected in the Sfold tool. Transfection of miRNA(s) in the Caco-2 cells, quantitatively estimated differential expression, confirming silencing of Tmprss2 with maximum gene suppression by hsa-miR-32 employing novel promising role in CoV-2 pathogenesis. The exalted binding of miRNAs to Tmprss2 and suppression of later advocates their utility as molecular tools for prevention of SARS-CoV-2 viral transmission and replication in humans.


Assuntos
MicroRNAs/metabolismo , Serina Endopeptidases/genética , Internalização do Vírus , Células CACO-2 , Biologia Computacional , Simulação por Computador , Inativação Gênica , Humanos , MicroRNAs/química , MicroRNAs/genética , Conformação de Ácido Nucleico
10.
Methods Mol Biol ; 2181: 253-267, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32729085

RESUMO

MicroRNAs (miRNAs) are a class of ~22 nt noncoding RNAs playing essential roles in the post-transcriptional regulation of gene expression, cell proliferation, and cell differentiation and are often found deregulated in several diseases including cancer.The A-to-I RNA editing, mediated by ADAR enzymes, is a diffuse post-transcriptional mechanism that converts the genetically coded adenosine (A) into inosine (I) at the RNA level. Among different RNA targets, the ADAR enzymes can also edit miRNA precursors. Specifically, a single nucleotide change (A/I) lying within the mature miRNA can alter the miRNA binding specificity and redirect the edited miRNA to a different mRNA target. In several cancer types a consistent deregulation of A-to-I RNA editing machinery also involves important miRNAs (either oncomiRs or tumor-suppressor miRNAs). Herein we describe a combined in silico and experimental approach for the detection of edited miRNAs and the identification and validation of their target genes potentially involved in cancer progression or invasion.


Assuntos
MicroRNAs/genética , Neoplasias/genética , Oncogenes , Edição de RNA/fisiologia , Análise de Sequência de DNA/métodos , Adenosina/análise , Adenosina/genética , Animais , Carcinogênese/genética , Biologia Computacional/métodos , Humanos , Inosina/análise , Inosina/genética , MicroRNAs/química , Neoplasias/patologia , Estudos de Validação como Assunto
11.
Methods Mol Biol ; 2211: 123-143, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33336275

RESUMO

The importance of peptide nucleic acids (PNAs) for alteration of gene expression is nowadays firmly established. PNAs are characterized by a pseudo-peptide backbone composed of N-(2-aminoethyl)glycine units and have been found to be excellent candidates for antisense and antigene therapies. Recently, PNAs have been demonstrated to alter the action of microRNAs and thus can be considered very important tools for miRNA therapeutics. In fact, the pharmacological modulation of microRNA activity appears to be a very interesting approach in the development of new types of drugs. Among the limits of PNAs in applied molecular biology, the delivery to target cells and tissues is of key importance. The aim of this chapter is to describe methods for the efficient delivery of unmodified PNAs designed to target microRNAs involved in cancer, using as model system miR-221-3p and human glioma cells as in vitro experimental cellular system. The methods employed to deliver PNAs targeting miR-221-3p here presented are based on a macrocyclic multivalent tetraargininocalix[4]arene used as non-covalent vector for anti-miR-221-3p PNAs. High delivery efficiency, low cytotoxicity, maintenance of the PNA biological activity, and easy preparation makes this vector a candidate for a universal delivery system for this class of nucleic acid analogs.


Assuntos
Portadores de Fármacos , Sistemas de Liberação de Medicamentos , Técnicas de Transferência de Genes , Ácidos Nucleicos Peptídicos/administração & dosagem , Apoptose , Linhagem Celular , Sobrevivência Celular/genética , Técnicas de Química Sintética , Humanos , MicroRNAs/administração & dosagem , MicroRNAs/química , MicroRNAs/genética , Estrutura Molecular , Ácidos Nucleicos Peptídicos/química
12.
Methods Mol Biol ; 2170: 53-77, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32797451

RESUMO

MicroRNAs control plant development and are key regulators of plant responses to biotic and abiotic stresses. Thus, their expression must be carefully controlled since both excess and deficiency of a given microRNA may be deleterious to plant cell. MicroRNA expression regulation can occur at several stages of their biogenesis pathway. One of the most important of these regulatory checkpoints is transcription efficiency. mirEX database is a tool for exploration and visualization of plant pri-miRNA expression profiles. It includes results obtained using high-throughput RT-qPCR platform designed to monitor pri-miRNA expression in different miRNA biogenesis mutants and developmental stages of Arabidopsis, barley, and Pellia plants. A step-by-step instruction for browsing the database and detailed protocol for high-throughput RT-qPCR experiments, including list of primers designed for the amplification of pri-miRNAs, are presented.


Assuntos
Arabidopsis/metabolismo , Hordeum/metabolismo , MicroRNAs/metabolismo , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Hordeum/genética , MicroRNAs/química
13.
Methods Mol Biol ; 2170: 117-124, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32797454

RESUMO

MicroRNAs (miRNAs) are small RNAs, that bind to mRNA targets and regulate their translation. Functional study of miRNAs and exploration of their utility as disease markers require miRNA extraction from biological samples, which contain large amounts of interfering compounds for downstream RNA identification and quantification. The most common extraction methods employ either silica columns or TRIzol reagent, but these approaches afford low recovery for small RNAs, possibly due to their short strand lengths. Here, we describe the fabrication of titanium dioxide nanofibers and the optimal extraction conditions to improve miRNA recovery from biological buffers, cell lysate, and serum.


Assuntos
Nanofibras/química , Titânio/química , Biomarcadores , Humanos , MicroRNAs/química
14.
Methods Mol Biol ; 2170: 125-131, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32797455

RESUMO

Next Generation Sequencing (NGS) is becoming a routine experimental technology. It has been a great success in recent years to profile small-RNA species using NGS. Indeed, a large quantity of small-RNA profiling data has been generated from NGS, and computational methods have been developed to process and analyze NGS data for the purpose of identification of novel and expressed small noncoding RNAs and analysis of their roles in nearly all biological processes and pathways in eukaryotes. We discuss here the computational procedures and major steps for identification of microRNAs and natural antisense transcript-originated small interfering RNAs (nat-siRNAs) from NGS small-RNA profiling data.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , MicroRNAs/química , RNA Interferente Pequeno/genética , Perfilação da Expressão Gênica , RNA de Cadeia Dupla/genética , RNA Interferente Pequeno/análise , Pequeno RNA não Traduzido/química , Análise de Sequência de RNA/métodos
15.
Proc Natl Acad Sci U S A ; 117(52): 33197-33203, 2020 12 29.
Artigo em Inglês | MEDLINE | ID: mdl-33318191

RESUMO

RNAs have important functions that are dictated by their structure. Indeed, small molecules that interact with RNA structures can perturb function, serving as chemical probes and lead medicines. Here we describe the development of a fragment-based approach to discover and optimize bioactive small molecules targeting RNA. We extended the target validation method chemical cross-linking and isolation by pull-down (Chem-CLIP) to identify and map the binding sites of low molecular weight fragments that engage RNA or Chem-CLIP fragment mapping (Chem-CLIP-Frag-Map). Using Chem-CLIP-Frag-Map, we identified several fragments that bind the precursor to oncogenic microRNA-21 (pre-miR-21). Assembly of these fragments provided a specific bioactive compound with improved potency that inhibits pre-miR-21 processing, reducing mature miR-21 levels. The compound exerted selective effects on the transcriptome and selectively mitigated a miR-21-associated invasive phenotype in triple-negative breast cancer cells. The Chem-CLIP-Frag-Map approach should prove general to expedite the identification and optimization of small molecules that bind RNA targets.


Assuntos
Antineoplásicos/química , Descoberta de Drogas/métodos , MicroRNAs/química , Bibliotecas de Moléculas Pequenas/química , Antineoplásicos/farmacologia , Linhagem Celular , Linhagem Celular Tumoral , Feminino , Humanos , Ligantes , MicroRNAs/metabolismo , Simulação de Acoplamento Molecular , Motivos de Nucleotídeos , Bibliotecas de Moléculas Pequenas/farmacologia , Neoplasias de Mama Triplo Negativas/metabolismo
16.
PLoS One ; 15(12): e0243319, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33320908

RESUMO

Here, we report a rapid and ultra-sensitive detection technique for fluorescent molecules called scanning single molecular counting (SSMC). The method uses a fluorescence-based digital measurement system to count single molecules in a solution. In this technique, noise is reduced by conforming the signal shape to the intensity distribution of the excitation light via a circular scan of the confocal region. This simple technique allows the fluorescent molecules to freely diffuse into the solution through the confocal region and be counted one by one and does not require statistical analysis. Using this technique, 28 to 62 aM fluorescent dye was detected through measurement for 600 s. Furthermore, we achieved a good signal-to-noise ratio (S/N = 2326) under the condition of 100 pM target nucleic acid by only mixing a hybridization-sensitive fluorescent probe, called Eprobe, into the target oligonucleotide solution. Combination of SSMC and Eprobe provides a simple, rapid, amplification-free, and high-sensitive target nucleic acid detection system. This method is promising for future applications to detect particularly difficult to design primers for amplification as miRNAs and other short oligo nucleotide biomarkers by only hybridization with high sensitivity.


Assuntos
Corantes Fluorescentes/química , MicroRNAs/análise , Oligonucleotídeos/química , Fluorescência , MicroRNAs/química , Hibridização de Ácido Nucleico
17.
Nat Commun ; 11(1): 5320, 2020 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-33087730

RESUMO

MicroRNAs (miRNAs) are endogenous small RNAs of ∼21 nt that regulate multiple biological pathways in multicellular organisms. They derive from longer transcripts that harbor an imperfect stem-loop structure. In plants, the ribonuclease type III DICER-LIKE1 assisted by accessory proteins cleaves the precursor to release the mature miRNA. Numerous studies highlight the role of the precursor secondary structure during plant miRNA biogenesis; however, little is known about the relevance of the precursor sequence. Here, we analyzed the sequence composition of plant miRNA primary transcripts and found specifically located sequence biases. We show that changes in the identity of specific nucleotides can increase or abolish miRNA biogenesis. Most conspicuously, our analysis revealed that the identity of the nucleotides at unpaired positions of the precursor plays a crucial role during miRNA biogenesis in Arabidopsis.


Assuntos
Arabidopsis/genética , Arabidopsis/metabolismo , MicroRNAs/biossíntese , MicroRNAs/genética , RNA de Plantas/biossíntese , RNA de Plantas/genética , Proteínas de Arabidopsis/metabolismo , Pareamento Incorreto de Bases , Proteínas de Ciclo Celular/metabolismo , Magnoliopsida/genética , Magnoliopsida/metabolismo , MicroRNAs/química , MicroRNAs/metabolismo , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Polimorfismo de Nucleotídeo Único , Processamento Pós-Transcricional do RNA , RNA de Plantas/química , Ribonuclease III/metabolismo
18.
Nature ; 584(7820): 279-285, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32760005

RESUMO

In pathophysiology, reactive oxygen species oxidize biomolecules that contribute to disease phenotypes1. One such modification, 8-oxoguanine2 (o8G), is abundant in RNA3 but its epitranscriptional role has not been investigated for microRNAs (miRNAs). Here we specifically sequence oxidized miRNAs in a rat model of the redox-associated condition cardiac hypertrophy4. We find that position-specific o8G modifications are generated in seed regions (positions 2-8) of selective miRNAs, and function to regulate other mRNAs through o8G•A base pairing. o8G is induced predominantly at position 7 of miR-1 (7o8G-miR-1) by treatment with an adrenergic agonist. Introducing 7o8G-miR-1 or 7U-miR-1 (in which G at position 7 is substituted with U) alone is sufficient to cause cardiac hypertrophy in mice, and the mRNA targets of o8G-miR-1 function in affected phenotypes; the specific inhibition of 7o8G-miR-1 in mouse cardiomyocytes was found to attenuate cardiac hypertrophy. o8G-miR-1 is also implicated in patients with cardiomyopathy. Our findings show that the position-specific oxidation of miRNAs could serve as an epitranscriptional mechanism to coordinate pathophysiological redox-mediated gene expression.


Assuntos
Cardiomegalia/genética , Cardiomegalia/patologia , Inativação Gênica , MicroRNAs/química , MicroRNAs/metabolismo , Animais , Pareamento de Bases , Linhagem Celular , Modelos Animais de Doenças , Guanina/análogos & derivados , Guanina/análise , Guanina/química , Guanina/metabolismo , Humanos , Camundongos , MicroRNAs/genética , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Oxirredução , Ratos , Transcrição Genética/genética , Transcriptoma/genética
19.
Int J Mol Sci ; 21(13)2020 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-32645951

RESUMO

The SARS-CoV-2 virus is a recently-emerged zoonotic pathogen already well adapted to transmission and replication in humans. Although the mutation rate is limited, recently introduced mutations in SARS-CoV-2 have the potential to alter viral fitness. In addition to amino acid changes, mutations could affect RNA secondary structure critical to viral life cycle, or interfere with sequences targeted by host miRNAs. We have analysed subsets of genomes from SARS-CoV-2 isolates from around the globe and show that several mutations introduce changes in Watson-Crick pairing, with resultant changes in predicted secondary structure. Filtering to targets matching miRNAs expressed in SARS-CoV-2-permissive host cells, we identified ten separate target sequences in the SARS-CoV-2 genome; three of these targets have been lost through conserved mutations. A genomic site targeted by the highly abundant miR-197-5p, overexpressed in patients with cardiovascular disease, is lost by a conserved mutation. Our results are compatible with a model that SARS-CoV-2 replication within the human host is constrained by host miRNA defences. The impact of these and further mutations on secondary structures, miRNA targets or potential splice sites offers a new context in which to view future SARS-CoV-2 evolution, and a potential platform for engineering conditional attenuation to vaccine development, as well as providing a better understanding of viral tropism and pathogenesis.


Assuntos
Betacoronavirus/genética , Genoma Viral , MicroRNAs/metabolismo , RNA Viral/química , Regiões 3' não Traduzidas , Sequência de Bases , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Bases de Dados Genéticas , Humanos , MicroRNAs/química , MicroRNAs/genética , Mutação , Conformação de Ácido Nucleico , Pandemias , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Sítios de Splice de RNA , Processamento de RNA , Alinhamento de Sequência , Proteínas não Estruturais Virais/genética , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismo
20.
Nucleic Acids Res ; 48(14): e80, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32496547

RESUMO

Small RNAs are important regulators of gene expression and are involved in human development and disease. Next generation sequencing (NGS) allows for scalable, genome-wide studies of small RNA; however, current methods are challenged by low sensitivity and high bias, limiting their ability to capture an accurate representation of the cellular small RNA population. Several studies have shown that this bias primarily arises during the ligation of single-strand adapters during library preparation, and that this ligation bias is magnified by 2'-O-methyl modifications (2'OMe) on the 3' terminal nucleotide. In this study, we developed a novel library preparation process using randomized splint ligation with a cleavable adapter, a design which resolves previous challenges associated with this ligation strategy. We show that a randomized splint ligation based workflow can reduce bias and increase the sensitivity of small RNA sequencing for a wide variety of small RNAs, including microRNA (miRNA) and tRNA fragments as well as 2'OMe modified RNA, including Piwi-interacting RNA and plant miRNA. Finally, we demonstrate that this workflow detects more differentially expressed miRNA between tumorous and matched normal tissues. Overall, this library preparation process allows for highly accurate small RNA sequencing and will enable studies of 2'OMe modified RNA with new levels of detail.


Assuntos
Biblioteca Gênica , Pequeno RNA não Traduzido/isolamento & purificação , Análise de Sequência de RNA/métodos , Eletroforese Capilar , Feminino , Humanos , Masculino , Metilação , MicroRNAs/química , MicroRNAs/genética , MicroRNAs/isolamento & purificação , Hibridização de Ácido Nucleico , Oligorribonucleotídeos/química , RNA Neoplásico/química , RNA Neoplásico/genética , RNA Neoplásico/isolamento & purificação , RNA de Plantas/química , RNA de Plantas/genética , RNA de Plantas/isolamento & purificação , Pequeno RNA não Traduzido/química , Pequeno RNA não Traduzido/genética , RNA de Transferência/química , RNA de Transferência/isolamento & purificação , Distribuição Aleatória , Sensibilidade e Especificidade , Alinhamento de Sequência
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