RESUMO
In cells, closely spaced microRNA (miRNA) target sites within a messenger RNA (mRNA) can act cooperatively, leading to more repression of the target mRNA than expected by independent action at each site. Using purified miRNA-Argonaute (AGO2) complexes, synthetic target RNAs, and a purified domain of TNRC6B (GW182 in flies) that is able to simultaneously bind multiple AGO proteins, we examined both the occupancies and binding affinities of miRNA-AGO2 complexes and target RNAs with either one site or two cooperatively spaced sites. On their own, miRNA-AGO2 complexes displayed little if any cooperative binding to dual sites. In contrast, in the presence of the AGO-binding region of TNRC6B, we observed strong cooperative binding to dual sites, with almost no singly bound target RNAs and substantially increased binding affinities and Hill coefficients. Cooperative binding was retained when the two sites were for two different miRNAs or when the two sites were bound to miRNAs loaded into two different AGO paralogs, AGO1 and AGO2. The improved binding affinity was attributable primarily to a reduced rate of dissociation between miRNA-AGO complexes and their dual-site targets. Thus, the multivalent binding of TNRC6 enables cooperative binding of miRNA-AGO complexes to target RNAs, thereby explaining the basis of cooperative action.
Assuntos
Regulação da Expressão Gênica , MicroRNAs/genética , Interferência de RNA , Proteínas Argonautas/metabolismo , Sítios de Ligação , Inativação Gênica , Humanos , Cinética , Modelos Biológicos , Ligação Proteica , Proteínas de Ligação a RNA/metabolismo , Complexo de Inativação Induzido por RNA/metabolismoRESUMO
MicroRNAs (miRNAs) are a large class of small noncoding RNAs that regulate the expression of distinct target mRNAs. miRNAs are incorporated into Argonaute (AGO) proteins and guide them to their target mRNAs. Subsequently, AGO proteins recruit a member of the glycine-tryptophan-rich (GW) protein family by direct protein-protein interaction. GW proteins coordinate all downstream processes leading to robust and efficient gene silencing. A short peptide of GW proteins comprising the AGO interaction motif can be used to biochemically isolate endogenous AGO protein complexes. Furthermore, within a cell such a peptide competes with endogenous GW proteins for AGO binding and thus can be used as potent inhibitor of the miRNA pathway. Here, we describe a method that utilizes a GW-based polypeptide (T6B-assay) to validate miRNA-mRNA interactions in tissue culture systems.
Assuntos
Proteínas Argonautas/metabolismo , Imunoprecipitação/métodos , MicroRNAs/antagonistas & inibidores , Peptídeos/uso terapêutico , Inativação Gênica/efeitos dos fármacos , MicroRNAs/genética , MicroRNAs/metabolismo , Peptídeos/metabolismo , Ligação Proteica , Mapas de Interação de Proteínas/genética , RNA Mensageiro/antagonistas & inibidoresRESUMO
During microRNA (miRNA)-guided gene silencing, Argonaute (Ago) proteins interact with a member of the TNRC6/GW protein family. Here we used a short GW protein-derived peptide fused to GST and demonstrate that it binds to Ago proteins with high affinity. This allows for the simultaneous isolation of all Ago protein complexes expressed in diverse species to identify associated proteins, small RNAs, or target mRNAs. We refer to our method as "Ago protein Affinity Purification by Peptides" (Ago-APP). Furthermore, expression of this peptide competes for endogenous TNRC6 proteins, leading to global inhibition of miRNA function in mammalian cells.
Assuntos
Proteínas Argonautas/isolamento & purificação , Cromatografia de Afinidade/métodos , Complexos Multiproteicos/isolamento & purificação , Peptídeos/isolamento & purificação , Sequência de Aminoácidos , Animais , Extratos Celulares , Precipitação Química , Drosophila melanogaster , Inativação Gênica , Células HEK293 , Células HeLa , Humanos , MicroRNAs/metabolismo , Dados de Sequência Molecular , Peptídeos/químicaRESUMO
MicroRNAs (miRNAs) are small evolutionarily conserved regulatory RNAs that modulate mRNA stability and translation in a wide range of cell types. MiRNAs are involved in a broad array of biological processes, including cellular proliferation, differentiation, and apoptosis. To identify previously unidentified regulators of miRNA, we initiated a systematic discovery-type proteomic analysis of the miRNA pathway interactome in human cells. Six of 66 genes identified in our proteomic screen were capable of regulating lethal-7a (let-7a) miRNA reporter activity. Tripartite motif 65 (TRIM65) was identified as a repressor of miRNA activity. Detailed analysis indicates that TRIM65 interacts and colocalizes with trinucleotide repeat containing six (TNRC6) proteins in processing body-like structures. Ubiquitination assays demonstrate that TRIM65 is an ubiquitin E3 ligase for TNRC6 proteins. The combination of overexpression and knockdown studies establishes that TRIM65 relieves miRNA-driven suppression of mRNA expression through ubiquitination and subsequent degradation of TNRC6.
