RESUMO
The UPF0054 protein family is highly conserved with homologues present in nearly every sequenced bacterium. In some bacteria, the respective gene is essential, while in others its loss results in a highly pleiotropic phenotype. Despite detailed structural studies, a cellular role for this protein family has remained unknown. We report here that deletion of the Escherichia coli homologue, YbeY, causes striking defects that affect ribosome activity, translational fidelity and ribosome assembly. Mapping of 16S, 23S and 5S rRNA termini reveals that YbeY influences the maturation of all three rRNAs, with a particularly strong effect on maturation at both the 5'- and 3'-ends of 16S rRNA as well as maturation of the 5'-termini of 23S and 5S rRNAs. Furthermore, we demonstrate strong genetic interactions between ybeY and rnc (encoding RNase III), ybeY and rnr (encoding RNase R), and ybeY and pnp (encoding PNPase), further suggesting a role for YbeY in rRNA maturation. Mutation of highly conserved amino acids in YbeY, allowed the identification of two residues (H114, R59) that were found to have a significant effect in vivo. We discuss the implications of these findings for rRNA maturation and ribosome assembly in bacteria.
Assuntos
Proteínas de Escherichia coli/metabolismo , Metaloproteínas/metabolismo , RNA Bacteriano/metabolismo , RNA Ribossômico/metabolismo , Sequência de Aminoácidos , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Deleção de Genes , Metaloproteínas/genética , Dados de Sequência Molecular , Fatores de Iniciação em Procariotos/metabolismo , Ligação Proteica , Ribossomos/metabolismo , Alinhamento de SequênciaRESUMO
The downregulation of many mRNAs has been observed through bioinformatic analysis of microarray results following transfection of short interfering RNAs (siRNAs). Many of these mRNA changes are due to the interaction of the siRNA guide strand with partially complementary sites and thus are considered "off-target" effects. To examine the mRNA:siRNA interactions important for off-target effects, we generated a panel of mRNA:siRNA combinations containing single and double mismatches, bulges, and noncanonical base-pairing interactions in the 9th, 10th, and 11th positions of two siRNA binding sites located in the 3' UTR of an integrated reporter gene. Approximately half of the mRNA:siRNA combinations containing mismatches in positions 9-11 result in a twofold or more mRNA decrease with varying degrees of protein knockdown. However, mRNA and protein analysis of the various mRNA:siRNA combinations reveals instances in which mRNA and protein levels do not correlate. Analysis of the resulting degradation products recovered from an imperfectly complementary siRNA interaction with an endogenous gene reveals a small fraction of products that map to the canonical siRNA cleavage site. Furthermore, downregulation of ARGONAUTE 2 (AGO2), the only AGO family protein known to catalyze canonical siRNA-mediated cleavage, did not significantly affect the degree of mRNA knockdown observed for one of the stably expressed reporters after transfection of an imperfectly complementary siRNA. These results indicate that although some degree of canonical siRNA cleavage can take place between a siRNA and an off-target transcript, most off-target mRNA reductions are likely attributable to AGO2-independent degradation processes.
Assuntos
Regulação da Expressão Gênica , Biossíntese de Proteínas/genética , Estabilidade de RNA , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Animais , Proteínas Argonautas , Sequência de Bases , Fator de Iniciação 2 em Eucariotos , Genes Reporter , Humanos , Luciferases de Renilla/análise , Luciferases de Renilla/genética , Dados de Sequência Molecular , Fatores de Iniciação de Peptídeos/antagonistas & inibidores , Fatores de Iniciação de Peptídeos/genética , Fatores de Iniciação de Peptídeos/metabolismo , RNA Mensageiro/antagonistas & inibidores , Transcrição GênicaRESUMO
Protein-protein interactions play a critical role in cellular processes such as signal transduction. Although many methods for identifying the binding partners of a protein of interest are available, it is currently difficult or impossible to assess the functional consequences of a specific interaction in vivo. To address this issue, we propose to modify proteins by addition of an artificial protein binding interface, thereby forcing them to interact in the cell in a pairwise fashion and allowing the functional consequences to be determined. For this purpose, we have developed an artificial binding interface consisting of a anti-Myc single-chain antibody (ScFv) and its peptide epitope. We found that the binding of an ScFv derived from anti-Myc monoclonal antibody 9E10 was relatively weak in vivo, so we selected an improved clone, 3DX, by in vitro mutagenesis and phage display. 3DX bound well to its epitope in a yeast two-hybrid system, and GST-fused 3DX also bound to several Myc-tagged proteins in mammalian cells. In vivo binding was relatively insensitive to the position of the ScFv in a fusion protein, but was improved by including multiple tandem copies of the Myc epitope in the binding partner. To test the system, we successfully replaced the SH3 domain-mediated interaction between the Abl tyrosine kinase and adaptor proteins Crk and Nck with an engineered interaction between 3DX and multiple Myc tags. We expect that this approach, which we term a functional interaction trap, will be a powerful proteomic tool for investigating protein-protein interactions.