RESUMO
Ultraviolet crosslinking and immunoprecipitation (CLIP) methodologies enable the identification of RNA binding sites of RNA-binding proteins (RBPs). Despite improvements in the library preparation of RNA fragments, the enhanced CLIP (eCLIP) protocol requires 4 days of hands-on time and lacks the ability to process several RBPs in parallel. We present a new method termed antibody-barcode eCLIP that utilizes DNA-barcoded antibodies and proximity ligation of the DNA oligonucleotides to RBP-protected RNA fragments to interrogate several RBPs simultaneously. We observe performance comparable with that of eCLIP with the advantage of dramatically increased scaling while maintaining the same material requirement of a single eCLIP experiment.
Assuntos
RNA , Transcriptoma , RNA/genética , Sítios de Ligação , Ligação Proteica , Proteínas de Ligação a RNA/metabolismo , Anticorpos/química , ImunoprecipitaçãoRESUMO
Transposable elements (TEs) occupy a large fraction of metazoan genomes and pose a constant threat to genomic integrity. This threat is particularly critical in germ cells, as changes in the genome that are induced by TEs will be transmitted to the next generation. Small noncoding piwi-interacting RNAs (piRNAs) recognize and silence a diverse set of TEs in germ cells. In mice, piRNA-guided transposon repression correlates with establishment of CpG DNA methylation on their sequences, yet the mechanism and the spectrum of genomic targets of piRNA silencing are unknown. Here we show that in addition to DNA methylation, the piRNA pathway is required to maintain a high level of the repressive H3K9me3 histone modification on long interspersed nuclear elements (LINEs) in germ cells. piRNA-dependent chromatin repression targets exclusively full-length elements of actively transposing LINE families, demonstrating the remarkable ability of the piRNA pathway to recognize active elements among the large number of genomic transposon fragments.
Assuntos
Células Germinativas/metabolismo , Elementos Nucleotídeos Longos e Dispersos/fisiologia , RNA Interferente Pequeno/metabolismo , Animais , Cromatina/metabolismo , Ilhas de CpG , Metilação de DNA , Regulação da Expressão Gênica , Inativação Gênica , Genoma/genética , Histonas/metabolismo , Elementos Nucleotídeos Longos e Dispersos/genética , Masculino , Camundongos , Mutação , RNA Interferente Pequeno/genéticaRESUMO
Although the functional parameters of microRNAs (miRNAs) have been explored in some depth, the roles of these molecules in viral infections remain elusive. Here we report a general method for global analysis of miRNA function that compares the significance of both overexpressing and inhibiting each mouse miRNA on the growth properties of different viruses. Our comparative analysis of representatives of all three herpesvirus subfamilies identified host miRNAs with broad anti- and proviral properties which extend to a single-stranded RNA virus. Specifically, we demonstrate the broad antiviral capacity of miR-199a-3p and illustrate that this individual host-encoded miRNA regulates multiple pathways required and/or activated by viruses, including PI3K/AKT and ERK/MAPK signaling, oxidative stress signaling, and prostaglandin synthesis. Global miRNA expression analysis further demonstrated that the miR-199a/miR-214 cluster is down-regulated in both murine and human cytomegalovirus infection and manifests similar antiviral properties in mouse and human cells. Overall, we report a general strategy for examining the contributions of individual host miRNAs in viral infection and provide evidence that these molecules confer broad inhibitory potential against multiple viruses.
Assuntos
Antivirais/análise , Estudo de Associação Genômica Ampla/métodos , Herpesviridae/efeitos dos fármacos , MicroRNAs/análise , Animais , Antivirais/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , MicroRNAs/farmacologia , Células NIH 3T3 , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND: Synapse formation and the development of neural networks are known to be controlled by a coordinated program of mRNA synthesis. microRNAs are now recognized to be important regulators of mRNA translation and stability in a wide variety of organisms. While specific microRNAs are known to be involved in neural development, the extent to which global microRNA and mRNA profiles are coordinately regulated in neural development is unknown. RESULTS: We examined mouse primary neuronal cultures, analyzing microRNA and mRNA expression. Three main developmental patterns of microRNA expression were observed: steady-state levels, up-regulated and down-regulated. Co-expressed microRNAs were found to have related target recognition sites and to be encoded in distinct genomic locations. A number of 43 differentially expressed miRNAs were located in five genomic clusters. Their predicted mRNA targets show reciprocal levels of expression. We identified a set of reciprocally expressed microRNAs that target mRNAs encoding postsynaptic density proteins and high-level steady-state microRNAs that target non-neuronal low-level expressed mRNAs. CONCLUSION: We characterized hundreds of miRNAs in neuronal culture development and identified three major modes of miRNA expression. We predict these miRNAs to regulate reciprocally expressed protein coding genes, including many genes involved in synaptogenesis. The identification of miRNAs that target mRNAs during synaptogenesis indicates a new level of regulation of the synapse.
Assuntos
Perfilação da Expressão Gênica , MicroRNAs/metabolismo , Neurogênese/genética , RNA Mensageiro/metabolismo , Animais , Análise por Conglomerados , Embrião de Mamíferos/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Genoma , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , Análise de Sequência de RNARESUMO
In developing male germ cells, prospermatogonia, two Piwi proteins, MILI and MIWI2, use Piwi-interacting RNA (piRNA) guides to repress transposable element (TE) expression and ensure genome stability and proper gametogenesis. In addition to their roles in post-transcriptional TE repression, both proteins are required for DNA methylation of TE sequences. Here, we analyzed the effect of Miwi2 deficiency on piRNA biogenesis and transposon repression. Miwi2 deficiency had only a minor impact on piRNA biogenesis; however, the piRNA profile of Miwi2-knockout mice indicated overexpression of several LINE1 TE families that led to activation of the ping-pong piRNA cycle. Furthermore, we found that MILI and MIWI2 have distinct functions in TE repression in the nucleus. MILI is responsible for DNA methylation of a larger subset of TE families than MIWI2 is, suggesting that the proteins have independent roles in establishing DNA methylation patterns.
Assuntos
Proteínas Argonautas/metabolismo , Metilação de DNA , RNA Interferente Pequeno/genética , Animais , Proteínas Argonautas/genética , Sequência de Bases , Elementos de DNA Transponíveis/genética , Elementos Nucleotídeos Longos e Dispersos/genética , Masculino , Camundongos , Dados de Sequência Molecular , Espermatogônias/metabolismoRESUMO
A single microRNA (miRNA) can inhibit a large number of mRNA transcripts. This widespread regulatory function has been experimentally demonstrated for a number of miRNAs. However, even when a multitude of targets is confirmed, function of a miRNA is frequently interpreted through a prism of a handful arbitrarily selected "interesting" targets. In this work we first show that hundreds of transcripts with target sites for two miRNAs expressed endogenously in neurons, miR-124 and miR-434-3p, are coordinately upregulated in a variety of neuronal stresses. This creates a landscape where these two miRNAs can exert their widespread inhibitory potential on stress-induced transcripts. Next, we experimentally demonstrate that overexpression of these two miRNAs indeed significantly inhibits expression of hundreds of stress-induced transcripts, thus confirming that these transcripts are enriched in true targets of examined miRNAs. A number of miRNAs were previously shown to have important roles in the regulation of stress responses, and our results suggest that these roles should be understood in light of a wide spread activation of miRNA targets during stresses. Importantly, a popular cationic lipid transfection reagent triggers such induction of miRNA targets. Therefore, when a transfection paradigm is employed to study miRNA function, the results of such studies should be interpreted with consideration for the inadvertent induction of miRNA targets.