RESUMO
Endonucleolytic cleavage of pre-mRNAs is the first step during eukaryotic mRNA 3' end formation. It has been proposed that cleavage factors CF IA, CF IB and CF II are required for pre-mRNA 3' end cleavage in yeast. CF IB is composed of a single polypeptide, Nab4p/Hrp1p, which is related to the A/B group of metazoan heterogeneous nuclear ribonucleoproteins (hnRNPs) that function as antagonistic regulators of 5' splice site selection. Here, we provide evidence that Nab4p/Hrp1p is not required for pre-mRNA 3' end endonucleolytic cleavage. We show that CF IA and CF II devoid of Nab4p/Hrp1p are sufficient to cleave a variety of RNA substrates but that cleavage occurs at multiple sites. Addition of Nab4p/Hrp1p prevents these alternative cleavages in a concentration-dependent manner, suggesting an essential and conserved role for some hnRNPs in pre-mRNA cleavage site selection.
Assuntos
Citocromos c , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA Fúngico/metabolismo , RNA Mensageiro/biossíntese , Ribonucleoproteínas/metabolismo , Proteínas de Saccharomyces cerevisiae , Sequência de Bases , Grupo dos Citocromos c/biossíntese , Grupo dos Citocromos c/genética , Ribonucleoproteínas Nucleares Heterogêneas , Modelos Genéticos , Dados de Sequência Molecular , Poli A/metabolismo , Proteínas de Ligação a RNA/metabolismo , Saccharomyces cerevisiae , Especificidade por Substrato , Fatores de Poliadenilação e Clivagem de mRNARESUMO
The Sendai virus nested set of C proteins which are expressed in an alternative open reading frame from the P mRNA has been shown to downregulate viral RNA synthesis. Utilizing a glutathione S-transferase (gst) C fusion protein (gstC), we have shown that C protein forms a complex with the L, but not the P, subunit of the viral RNA polymerase. When P, L, and gstC are coexpressed, an oligomer of P, through its interaction with L, is also bound to beads. Since binding of C to L in the P-L complex does not disrupt P binding, the C and P binding sites appear to be different. GstC binding to L occurs only when the proteins are coexpressed in the same cell. The gstC, but not gst, protein inhibits viral transcription in vitro, showing that the fusion protein retains biological function. Pulse-chase experiments of the various complexes show that L protein synthesized alone has a half-life of 1. 2 hr, which is increased 12.5-fold by binding P, but is not significantly increased by binding gstC. Analyses of complex formation with truncations of L protein show that the C-terminal 1333 amino acids of L are not required for binding C. The dose-response curves show that replication of the genomic DI-H RNA is more sensitive to inhibition by C protein than is the synthesis of DI leader RNA, suggesting that the downregulation of RNA synthesis may be more complex than just the inhibition of the initiation of RNA synthesis.
Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Regulação Viral da Expressão Gênica , Nucleoproteínas , Proteínas Virais/metabolismo , Animais , Sítios de Ligação/genética , Chlorocebus aethiops , Glutationa Transferase , Humanos , Proteínas do Nucleocapsídeo , Fosfoproteínas/metabolismo , Plasmídeos , RNA Mensageiro/análise , RNA Viral/antagonistas & inibidores , RNA Viral/biossíntese , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/fisiologia , Transcrição Gênica , Transfecção , Células Vero , Proteínas do Core Viral/genética , Proteínas Virais/genética , Proteínas Virais/fisiologiaRESUMO
A 21-year-old man developed blurred vision and retinal pigment epithelium changes similar to those in acute posterior multifocal placoid pigment epitheliopathy following vaccination for swine flu. The patient's medical examination revealed no infectious agents. A hypersensitivity to the attenuated virus of the swine flu vaccine appears to have produced his initial flu-like symptoms and retinal pigment epithelium changes.
