RESUMO
Cell biologists have long speculated that a minus end-directed motor localized at kinetochores contributes to the poleward movement of chromosomes during mitosis. Two recent studies provide direct evidence that cytoplasmic dynein can perform this function.
Assuntos
Proteínas de Ciclo Celular , Cromossomos/fisiologia , Proteínas de Drosophila , Dineínas/fisiologia , Cinetocoros/fisiologia , Mitose/fisiologia , Proteínas de Xenopus , Animais , Proteína Quinase CDC2/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Ciclina B/metabolismo , Complexo Dinactina , Dineínas/metabolismo , Proteínas de Insetos/metabolismo , Cinesinas/metabolismo , Cinetocoros/metabolismo , Meiose/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Fuso Acromático/metabolismo , Fuso Acromático/fisiologiaRESUMO
We previously identified a 160-nucleotide packaging signal, MPsi, from the 5' end of the Rous sarcoma virus genome. In this study, we determine the secondary structure of MPsi by using phylogenetic analysis with computer modeling and heterologous packaging assays of point mutants. The results of the in vivo studies are in good agreement with the computer model. Additionally, the packaging studies indicate several structures which are important for efficient packaging, including a single-stranded bulge containing the initiation codon for the short open reading frame, uORF3, as well as adjacent stem structures. Finally, we show that the L3 stem-loop at the 3' end of MPsi is dispensable for packaging, thus identifying an 82-nucleotide minimal packaging signal, microPsi, composed of the O3 stem-loop.
Assuntos
Alpharetrovirus/fisiologia , Conformação de Ácido Nucleico , RNA Viral/química , Montagem de Vírus , Alpharetrovirus/genética , Sequência de Bases , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fases de Leitura Aberta , Filogenia , RNA Viral/genética , Homologia de Sequência do Ácido NucleicoRESUMO
Retroviruses preferentially package full-length genomic RNA over spliced viral messages. For most retroviruses, this preference is likely due to the absence of all or part of the packaging signal on subgenomic RNAs. In avian leukosis-sarcoma virus, however, we have shown that the minimal packaging signal, MPsi, is located upstream of the 5' splice site and therefore is present on both genomic and spliced RNAs. We now show that an MPsi-containing heterologous RNA is packaged only 2.6-fold less efficiently than genomic Rous sarcoma virus RNA. Thus, few additional packaging sequences and/or structures exist outside of MPsi. In contrast, we found that env mRNA is not efficiently packaged. These results indicate that either MPsi is not functional on this RNA or the RNA is somehow segregated from the packaging machinery. Finally, deletion of sequences from the 3' end of MPsi was found to reduce the packaging efficiency of heterologous RNAs.
Assuntos
Alpharetrovirus/metabolismo , Vírus do Sarcoma Aviário/metabolismo , Produtos do Gene env/genética , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Montagem de Vírus , Sequência de Bases , Linhagem Celular , Produtos do Gene env/biossíntese , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Plasmídeos , Splicing de RNA , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Viral/química , RNA Viral/genética , Proteínas Virais/análiseRESUMO
We have defined a 160-nucleotide region, Mpsi, from the 5' leader region of the Rous sarcoma virus genome that is sufficient to direct the packaging of a heterologous RNA. Mpsi contains the putative O3 stem structure that has previously been shown, and that has been confirmed in this study, to be important for the efficient packaging of avian leukosis-sarcoma virus RNA. Analyses of several O3 stem mutants revealed that other regions within Mpsi can interfere with the proper folding of altered sequences which are predicted to form a wild-type O3 stem.
