RESUMO
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.
Assuntos
MicroRNAs/metabolismo , Processamento Pós-Transcricional do RNA , Ribonuclease III/química , Sequência de Aminoácidos , Cristalografia por Raios X , RNA Helicases DEAD-box/química , RNA Helicases DEAD-box/metabolismo , Evolução Molecular , Humanos , MicroRNAs/química , Modelos Químicos , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Dobramento de Proteína , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA/metabolismo , Ribonuclease III/genética , Ribonuclease III/metabolismo , Alinhamento de Sequência , Homologia Estrutural de ProteínaRESUMO
MicroRNA (miRNA) maturation is initiated by Microprocessor composed of RNase III DROSHA and its cofactor DGCR8, whose fidelity is critical for generation of functional miRNAs. To understand how Microprocessor recognizes pri-miRNAs, we here reconstitute human Microprocessor with purified recombinant proteins. We find that Microprocessor is an â¼364 kDa heterotrimeric complex of one DROSHA and two DGCR8 molecules. Together with a 23-amino acid peptide from DGCR8, DROSHA constitutes a minimal functional core. DROSHA serves as a "ruler" by measuring 11 bp from the basal ssRNA-dsRNA junction. DGCR8 interacts with the stem and apical elements through its dsRNA-binding domains and RNA-binding heme domain, respectively, allowing efficient and accurate processing. DROSHA and DGCR8, respectively, recognize the basal UG and apical UGU motifs, which ensure proper orientation of the complex. These findings clarify controversies over the action mechanism of DROSHA and allow us to build a general model for pri-miRNA processing.
Assuntos
MicroRNAs/metabolismo , Processamento Pós-Transcricional do RNA , Proteínas de Ligação a RNA/química , Ribonuclease III/química , Sequência de Bases , Dimerização , Humanos , MicroRNAs/genética , Dados de Sequência Molecular , Motivos de Nucleotídeos , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribonuclease III/genética , Ribonuclease III/metabolismoRESUMO
Microprocessor, composed of DROSHA and its cofactor DGCR8, initiates microRNA (miRNA) biogenesis by processing the primary transcripts of miRNA (pri-miRNAs). Here we investigate the mechanism by which Microprocessor selects the cleavage site with single-nucleotide precision, which is crucial for the specificity and functionality of miRNAs. By testing â¼40,000 pri-miRNA variants, we find that for some pri-miRNAs the cleavage site is dictated mainly by the mGHG motif embedded in the lower stem region of pri-miRNA. Structural modeling and deep-sequencing-based complementation experiments show that the double-stranded RNA-binding domain (dsRBD) of DROSHA recognizes mGHG to place the catalytic center in the appropriate position. The mGHG motif as well as the mGHG-recognizing residues in DROSHA dsRBD are conserved across eumetazoans, suggesting that this mechanism emerged in an early ancestor of the animal lineage. Our findings provide a basis for the understanding of miRNA biogenesis and rational design of accurate small-RNA-based gene silencing.
Assuntos
MicroRNAs/metabolismo , Motivos de Nucleotídeos , Processamento Pós-Transcricional do RNA , Ribonuclease III/metabolismo , Células HCT116 , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , MicroRNAs/química , MicroRNAs/genética , Modelos Moleculares , Conformação de Ácido Nucleico , Domínios e Motivos de Interação entre Proteínas , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribonuclease III/genética , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Microprocessor, which consists of a ribonuclease III DROSHA and its cofactor DGCR8, initiates microRNA (miRNA) maturation by cleaving primary miRNA transcripts (pri-miRNAs). We recently demonstrated that the DGCR8 dimer recognizes the apical elements of pri-miRNAs, including the UGU motif, to accurately locate and orient Microprocessor on pri-miRNAs. However, the mechanism underlying the selective RNA binding remains unknown. In this study, we find that hemin, a ferric ion-containing porphyrin, enhances the specific interaction between the apical UGU motif and the DGCR8 dimer, allowing Microprocessor to achieve high efficiency and fidelity of pri-miRNA processing in vitro. Furthermore, by generating a DGCR8 mutant cell line and carrying out rescue experiments, we discover that hemin preferentially stimulates the expression of miRNAs possessing the UGU motif, thereby conferring differential regulation of miRNA maturation. Our findings reveal the molecular action mechanism of hemin in pri-miRNA processing and establish a novel function of hemin in inducing specific RNA-protein interaction.
Assuntos
Hemina/fisiologia , MicroRNAs/metabolismo , Proteínas de Ligação a RNA/metabolismo , Linhagem Celular , Técnicas de Inativação de Genes , Humanos , MicroRNAs/química , Precursores de RNA/química , Precursores de RNA/metabolismo , Proteínas de Ligação a RNA/genética , Ribonuclease III/metabolismoRESUMO
p62/SQSTM1 is known to act as a key mediator in the selective autophagy of protein aggregates, or aggrephagy, by steering ubiquitinated protein aggregates towards the autophagy pathway. Here, we use a yeast two-hybrid screen to identify the prefoldin-like chaperone UXT as an interacting protein of p62. We show that UXT can bind to protein aggregates as well as the LB domain of p62, and, possibly by forming an oligomer, increase p62 clustering for its efficient targeting to protein aggregates, thereby promoting the formation of the p62 body and clearance of its cargo via autophagy. We also find that ectopic expression of human UXT delays SOD1(A4V)-induced degeneration of motor neurons in a Xenopus model system, and that specific disruption of the interaction between UXT and p62 suppresses UXT-mediated protection. Together, these results indicate that UXT functions as an autophagy adaptor of p62-dependent aggrephagy. Furthermore, our study illustrates a cooperative relationship between molecular chaperones and the aggrephagy machinery that efficiently removes misfolded protein aggregates.
Assuntos
Autofagia/genética , Proteínas de Ciclo Celular/genética , Chaperonas Moleculares/genética , Agregados Proteicos , Proteína Sequestossoma-1/genética , Superóxido Dismutase-1/genética , Animais , Autofagia/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Gânglios Espinais/citologia , Gânglios Espinais/metabolismo , Regulação da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Células HeLa , Humanos , Leupeptinas/farmacologia , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Chaperonas Moleculares/metabolismo , Neurônios Motores/citologia , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/metabolismo , Cultura Primária de Células , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Agregados Proteicos/efeitos dos fármacos , Dobramento de Proteína/efeitos dos fármacos , Proteína Sequestossoma-1/metabolismo , Transdução de Sinais , Superóxido Dismutase-1/metabolismo , Transgenes , Xenopus laevis , Proteína Vermelha FluorescenteRESUMO
Selective recognition and elimination of misfolded polypeptides are crucial for protein homeostasis. When the ubiquitin-proteasome system is impaired, misfolded polypeptides tend to form small cytosolic aggregates and are transported to the aggresome and eventually eliminated by the autophagy pathway. Despite the importance of this process, the regulation of aggresome formation remains poorly understood. Here, we identify TRIM28/TIF1ß/KAP1 (tripartite motif containing 28) as a negative regulator of aggresome formation. Direct interaction between TRIM28 and CTIF (cap binding complex dependent translation initiation factor) leads to inefficient aggresomal targeting of misfolded polypeptides. We also find that either treatment of cells with poly I:C or infection of the cells by influenza A viruses triggers the phosphorylation of TRIM28 at S473 in a way that depends on double-stranded RNA-activated protein kinase. The phosphorylation promotes association of TRIM28 with CTIF, inhibits aggresome formation, and consequently suppresses viral proliferation. Collectively, our data provide compelling evidence that TRIM28 is a negative regulator of aggresome formation.Abbreviations: BAG3: BCL2-associated athanogene 3; CTIF: CBC-dependent translation initiation factor; CED: CTIF-EEF1A1-DCTN1; DCTN1: dynactin subunit 1; EEF1A1: eukaryotic translation elongation factor 1 alpha 1; EIF2AK2: eukaryotic translation initiation factor 2 alpha kinase 2; HDAC6: histone deacetylase 6; IAV: influenza A virus; IP: immunoprecipitation; PLA: proximity ligation assay; polypeptidyl-puro: polypeptidyl-puromycin; qRT-PCR: quantitative reverse-transcription PCR; siRNA: small interfering RNA.
Assuntos
Autofagia , Vírus da Influenza A , Corpos de Inclusão/metabolismo , Vírus da Influenza A/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismoRESUMO
The overproduction and purification of human proteins is a requisite of both basic and medical research. Although many recombinant human proteins have been purified, current protein production methods have several limitations; recombinant proteins are frequently truncated, fail to fold properly, and/or lack appropriate post-translational modifications. In addition, such methods require subcloning of the target gene into relevant plasmids, which can be difficult for long proteins with repeated domains. Here we devised a novel method for target protein production by introduction of a strong promoter for overexpression and an epitope tag for purification in front of the endogenous human gene, in a sense performing molecular cloning directly in the human genome, which does not require cloning of the target gene. As a proof of concept, we successfully purified intact human Reelin protein, which is lengthy (3460 amino acids) and contains repeating domains, and confirmed that it was biologically functional.
Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Edição de Genes/métodos , Proteínas do Tecido Nervoso/metabolismo , Serina Endopeptidases/metabolismo , Sistemas CRISPR-Cas/genética , Moléculas de Adesão Celular Neuronais/análise , Moléculas de Adesão Celular Neuronais/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Proteínas da Matriz Extracelular/análise , Proteínas da Matriz Extracelular/genética , Humanos , Microscopia de Fluorescência , Proteínas do Tecido Nervoso/análise , Proteínas do Tecido Nervoso/genética , Plasmídeos/genética , Plasmídeos/metabolismo , RNA Guia de Cinetoplastídeos/metabolismo , Proteínas Recombinantes/análise , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Proteína Reelina , Serina Endopeptidases/análise , Serina Endopeptidases/genética , Espectrometria de Massas em TandemRESUMO
Mice were fed either 13 nm silver nanoparticles or 2-3.5 mum silver microparticles. The livers were then obtained after 3 days and subjected to a histopathological analysis. The nanoparticle-fed and microparticle-fed livers both exhibited lymphocyte infiltration in the histopathological analysis, suggesting the induction of inflammation. In vitro, a human hepatoma cell line (Huh-7) was treated with the same silver nanoparticles and microparticles. The mitochondrial activity and glutathione production were hardly affected. However, the DNA contents decreased 15% in the nanoparticle-treated cells and 10% in the microparticle-treated cell, suggesting a more potent induction of apoptosis by the nanoparticles. From a microarray analysis of the RNA from the livers of the nano- and micro-particle-fed mice, the expression of genes related to apoptosis and inflammation was found to be altered. These gene expression changes in the nanoparticle-treated livers lead to phenotypical changes, reflecting increased apoptosis and inflammation. The changes in the gene expression were confirmed by using a semi-quantitative RT-PCR.