RESUMO
Argonaute (Ago) proteins interact with small regulatory RNAs such as microRNAs (miRNAs) and facilitate gene-silencing processes. miRNAs guide Ago proteins to specific mRNAs leading to translational silencing or mRNA decay. In order to understand the mechanistic details of miRNA function, it is important to characterize Ago protein interactors. Although several proteomic studies have been performed, it is not clear how the Ago interactome changes on miRNA or mRNA binding. Here, we report the analysis of Ago protein interactions in miRNA-containing and miRNA-depleted cells. Using stable isotope labeling in cell culture in conjunction with Dicer knock out mouse embryonic fibroblasts, we identify proteins that interact with Ago2 in the presence or the absence of Dicer. In contrast to our current view, we find that Ago-mRNA interactions can also take place in the absence of miRNAs. Our proteomics approach provides a rich resource for further functional studies on the cellular roles of Ago proteins.
Assuntos
Proteínas Argonautas/metabolismo , RNA Helicases DEAD-box/metabolismo , Fibroblastos/metabolismo , Mamíferos/metabolismo , Mapas de Interação de Proteínas , Ribonuclease III/metabolismo , Animais , Western Blotting , Embrião de Mamíferos/citologia , Camundongos , MicroRNAs/metabolismo , Ligação Proteica , Reprodutibilidade dos Testes , Ribonucleoproteínas/metabolismoRESUMO
Murine gammaherpesvirus 68 (MHV-68) is closely related to Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) and provides a small-animal model with which to study the pathogenesis of gammaherpesvirus (gammaHV) infections. To completely explore the potential of the MHV-68 system for the investigation of gammaHV microRNAs (miRNAs), it would be desirable to know the number and expression patterns of all miRNAs encoded by MHV-68. By deep sequencing of small RNAs, we systematically investigated the expression profiles of MHV-68 miRNAs in both lytically and persistently infected cells. In addition to the nine known MHV-68 miRNAs, we identified six novel MHV-68 miRNA genes and analyzed the expression levels of all MHV-68 miRNAs. Furthermore, we also characterized the cellular miRNA expression signatures in MHV-68-infected versus noninfected NIH 3T3 fibroblasts and in 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-treated versus nontreated S11 cells. We found that mmu-mir-15b and mmu-mir-16 are highly upregulated upon MHV-68 infection of NIH 3T3 cells, indicating a potential role for cellular miRNAs during MHV-68 infection. Our data will aid in the full exploration of the functions of gammaHV miRNAs.
Assuntos
MicroRNAs/genética , RNA Viral/genética , Rhadinovirus/genética , Animais , Sequência de Bases , Linhagem Celular , Primers do DNA/genética , Perfilação da Expressão Gênica , Genoma Viral , Interações Hospedeiro-Patógeno/genética , Camundongos , MicroRNAs/química , Células NIH 3T3 , Conformação de Ácido Nucleico , RNA Viral/química , Rhadinovirus/patogenicidade , Rhadinovirus/fisiologia , Análise de Sequência de RNARESUMO
Azidothymidine (AZT, zidovudine) is one of the few nucleoside inhibitors known to inhibit foamy virus replication. We have shown previously that up to four mutations in the reverse transcriptase gene of simian foamy virus from macaque (SFVmac) are necessary to confer high resistance against AZT. To characterize the mechanism of AZT resistance we expressed two recombinant reverse transcriptases of highly AZT-resistant SFVmac in Escherichia coli harboring three (K211I, S345T, E350K) or four mutations (K211I, I224T, S345T, E350K) in the reverse transcriptase gene. Our analyses show that the polymerization activity of these mutants is impaired. In contrast to the AZT-resistant reverse transcriptase of HIV-1, the AZT resistant enzymes of SFVmac reveal differences in their kinetic properties. The SFVmac enzymes exhibit lower specific activities on poly(rA)/oligo(dT) and higher K(M)-values for polymerization but no change in K(D)-values for DNA/DNA or RNA/DNA substrates. The AZT resistance of the mutant enzymes is based on the excision of the incorporated inhibitor in the presence of ATP. The additional amino acid change of the quadruple mutant appears to be important for regaining polymerization efficiency.
Assuntos
Trifosfato de Adenosina/metabolismo , Didesoxinucleotídeos/metabolismo , DNA Polimerase Dirigida por RNA/genética , DNA Polimerase Dirigida por RNA/metabolismo , Inibidores da Transcriptase Reversa/farmacologia , Spumavirus/enzimologia , Nucleotídeos de Timina/metabolismo , Zidovudina/análogos & derivados , Zidovudina/farmacologia , Sequência de Aminoácidos , DNA/biossíntese , Farmacorresistência Viral/genética , Escherichia coli/genética , Cinética , Dados de Sequência Molecular , Mutação , RNA , DNA Polimerase Dirigida por RNA/química , Alinhamento de Sequência , Spumavirus/efeitos dos fármacos , Zidovudina/metabolismoRESUMO
Presequence protease PreP is a novel protease that degrades targeting peptides as well as other unstructured peptides in both mitochondria and chloroplasts. The first structure of PreP from Arabidopsis thaliana refined at 2.1 Angstroms resolution shows how the 995-residue polypeptide forms a unique proteolytic chamber of more than 10,000 Angstroms(3) in which the active site resides. Although there is no visible opening to the chamber, a peptide is bound to the active site. The closed conformation places previously unidentified residues from the C-terminal domain at the active site, separated by almost 800 residues in sequence to active site residues located in the N-terminal domain. Based on the structure, a novel mechanism for proteolysis is proposed involving hinge-bending motions that cause the protease to open and close in response to substrate binding. In support of this model, cysteine double mutants designed to keep the chamber covalently locked show no activity under oxidizing conditions. The manner in which substrates are processed inside the chamber is reminiscent of the proteasome; therefore, we refer to this protein as a peptidasome.