RESUMO
The RNA-binding protein Y14 binds preferentially to mRNAs produced by splicing and is a component of a multiprotein complex that assembles approximately 20 nucleotides upstream of exon-exon junctions. This complex probably has important functions in post-splicing events including nuclear export and nonsense-mediated decay of mRNA. We show that Y14 binds to two previously reported components, Aly/REF and RNPS1, and to the mRNA export factor TAP. Moreover, we identified magoh, a human homolog of the Drosophila mago nashi gene product, as a novel component of the complex. Magoh binds avidly and directly to Y14 and TAP, but not to other known components of the complex, and is found in Y14-containing mRNPs in vivo. Importantly, magoh also binds to mRNAs produced by splicing upstream (approximately 20 nucleotides) of exon- exon junctions and its binding to mRNA persists after export. These experiments thus reveal specific protein-protein interactions among the proteins of the splicing-dependent mRNP complex and suggest an important role for the highly evolutionarily conserved magoh protein in this complex.
Assuntos
Proteínas de Drosophila , Éxons , Proteínas Nucleares/metabolismo , Proteínas Nucleares/fisiologia , Splicing de RNA , Animais , Fracionamento Celular , Linhagem Celular , Núcleo Celular/metabolismo , Citoplasma/metabolismo , DNA Complementar/metabolismo , Drosophila , Evolução Molecular , Glutationa Transferase/metabolismo , Células HeLa , Humanos , Microscopia de Fluorescência , Modelos Biológicos , Oócitos/metabolismo , Plasmídeos/metabolismo , Testes de Precipitina , Ligação Proteica , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA , Proteínas Recombinantes de Fusão/metabolismo , Ribonuclease H/metabolismo , Técnicas do Sistema de Duplo-Híbrido , XenopusRESUMO
We recently described an RNA-binding protein, Y14, that binds preferentially to spliced mRNAs and persists in the cytoplasm. Y14 is part of a multi-protein complex that also contains the mRNA export factor TAP. This suggests that splicing imprints the mRNA with a unique set of proteins that communicate the history of the transcript to the cytoplasm. Here, using microinjection of pre-mRNAs into Xenopus oocyte nuclei followed by immunoprecipitation of RNase-fragmented mRNAs from the cytoplasm, we show that Y14 is stably bound to sequences immediately upstream of exon-exon junctions. This feature appears to be unique to Y14. Using monoclonal antibodies that we produced against Aly/REF, another component recently reported to be an mRNA export factor, we show that Aly/REF is associated with spliced mRNAs in the nucleus but is not detectable on mRNAs in the cytoplasm. Thus, we propose that the splicing- dependent binding of Y14 provides a position-specific molecular memory that communicates to the cytoplasm the location of exon and intron boundaries. This novel mechanism may play an important role in post-splicing events.
Assuntos
Núcleo Celular/metabolismo , Citoplasma/metabolismo , Éxons , Splicing de RNA , Proteínas de Ligação a RNA/metabolismo , Transporte Ativo do Núcleo Celular , Sítios de Ligação , Modelos Biológicos , Fatores de Transcrição/metabolismoRESUMO
AG7088 is a potent, irreversible inhibitor of human rhinovirus (HRV) 3C protease (inactivation rate constant (k(obs)/[I]) = 1,470,000 +/- 440,000 M(-1) s(-1) for HRV 14) that was discovered by protein structure-based drug design methodologies. In H1-HeLa and MRC-5 cell protection assays, AG7088 inhibited the replication of all HRV serotypes (48 of 48) tested with a mean 50% effective concentration (EC(50)) of 0.023 microM (range, 0.003 to 0.081 microM) and a mean EC(90) of 0.082 microM (range, 0.018 to 0.261 microM) as well as that of related picornaviruses including coxsackieviruses A21 and B3, enterovirus 70, and echovirus 11. No significant reductions in the antiviral activity of AG7088 were observed when assays were performed in the presence of alpha(1)-acid glycoprotein or mucin, proteins present in nasal secretions. The 50% cytotoxic concentration of AG7088 was >1,000 microM, yielding a therapeutic index of >12,346 to >333,333. In a single-cycle, time-of-addition assay, AG7088 demonstrated antiviral activity when added up to 6 h after infection. In contrast, a compound targeting viral attachment and/or uncoating was effective only when added at the initiation of virus infection. Direct inhibition of 3C proteolytic activity in infected cells treated with AG7088 was demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of radiolabeled proteins, which showed a dose-dependent accumulation of viral precursor polyproteins and reduction of processed protein products. The broad spectrum of antiviral activity of AG7088, combined with its efficacy even when added late in the virus life cycle, highlights the advantages of 3C protease as a target and suggests that AG7088 will be a promising clinical candidate.