RESUMO
One important function of the human adenovirus E1B 55-kDa protein is induction of selective nuclear export of viral late mRNAs. This protein interacts with the viral E4 Orf6 and four cellular proteins to form an infected-cell-specific E3 ubiquitin ligase. The assembly of this enzyme is required for efficient viral late mRNA export, but neither the relevant substrates nor the cellular pathway that exports viral late mRNAs has been identified. We therefore examined the effects on viral late gene expression of inhibition of the synthesis or activity of the mRNA export receptor Nxf1, which was observed to colocalize with the E1B 55-kDa protein in infected cells. When production of Nxf1 was impaired by using RNA interference, the efficiency of viral late mRNA export was reduced to a corresponding degree. Furthermore, synthesis of a dominant-negative derivative of Nxf1 during the late phase of infection interfered with production of a late structural protein. These observations indicate that the Nxf1 pathway is responsible for export of viral late mRNAs. As the infected-cell-specific E3 ubiquitin ligase targets its known substrates for proteasomal degradation, we compared the concentrations of several components of this pathway (Nxf1, Thox1, and Thoc4) in infected cells that did or did not contain this enzyme. Although the concentration of a well-established substrate, Mre11, decreased significantly in cells infected by adenovirus type 5 (Ad5), but not in those infected by the E1B 55-kDa protein-null mutant Hr6, no E1B 55-kDa protein-dependent degradation of the Nxf1 pathway proteins was observed.
Assuntos
Adenovírus Humanos/metabolismo , Núcleo Celular/metabolismo , Regulação Viral da Expressão Gênica , Proteínas de Transporte Nucleocitoplasmático/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transporte Ativo do Núcleo Celular , Proteínas E1B de Adenovirus/genética , Proteínas E1B de Adenovirus/metabolismo , Proteínas E1B de Adenovirus/fisiologia , Proteínas E4 de Adenovirus/genética , Proteínas E4 de Adenovirus/fisiologia , Adenovírus Humanos/genética , Células HeLa , Humanos , Proteínas de Transporte Nucleocitoplasmático/genética , Interferência de RNA , RNA Mensageiro/genética , RNA Viral/genética , RNA Viral/metabolismo , Proteínas de Ligação a RNA/genéticaRESUMO
The signaling and adaptor protein Homer3 plays a role in controlling immune homeostasis and self-reactivity. Homer3 is recruited to the immune synapse (IS) following TCR ligation, although the mechanisms regulating this subcellular localization are unknown. We show that Homer3 specifically associates with a novel ubiquitin-like domain in the IkappaB kinase (IKK) beta subunit of the IKK complex. Homer3 associates with IKKbeta in T cells and colocalizes with the IKK complex at the IS. However, Homer3 is not required for IKK activation, as NF-kappaB signaling is intact in Homer3-deficient T cells. Instead, the IKK complex recruits Homer3 to the IS following TCR engagement, and we present evidence that this association regulates actin dynamics in T cells. These findings identify a novel interaction between two major signaling proteins and reveal an unexpected NF-kappaB-independent function for the IKK complex in regulating the subcellular localization of Homer3.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas I-kappa B/metabolismo , Proteínas de Transporte/fisiologia , Linhagem Celular , Células HeLa , Proteínas de Arcabouço Homer , Humanos , Proteínas I-kappa B/fisiologia , Sinapses Imunológicas/enzimologia , Sinapses Imunológicas/imunologia , Células Jurkat , Estrutura Terciária de Proteína , Receptores de Antígenos de Linfócitos T/fisiologia , Transdução de Sinais/imunologia , Frações Subcelulares/imunologia , Frações Subcelulares/metabolismo , Ubiquitina/química , Ubiquitina/metabolismoRESUMO
A major rate-limiting step in transcription initiation by RNA polymerase II is recognition and binding of the TATA element by the transcription factor TFIID. TFIID is composed of TATA binding protein (TBP) and approximately a dozen TBP-associated factors (TAFs). Emerging consensus regarding the role of TAFs is that TFIID assumes a gene specific activity that is regulated by interaction with other factors. In spite of many studies demonstrating the essential nature of TAFs in transcription, very little is known about the subunit contacts within TFIID. To understand fully the functional role of TAFs, it is imperative to define TAF-TAF interactions and their topological arrangement within TFIID. We performed a systematic two-hybrid analysis using the 13 essential TAFs of the Saccharomyces cerevisiae TFIID complex and TBP. Specific interactions were defined for each component, and the biological significance of these interactions is supported by numerous genetic and biochemical studies. By combining the interaction profiles presented here, and the available studies utilizing specific TAFs, we propose a working hypothesis for the arrangement of components in the TFIID complex. Thus, these results serve as a foundation for understanding the overall architecture of yeast TFIID.