RESUMO
Cajal bodies (CBs) are nuclear organelles that are usually identified by the marker protein p80-coilin. Because no orthologue of coilin is known in Drosophila melanogaster, we identified D. melanogaster CBs using probes for other components that are relatively diagnostic for CBs in vertebrate cells. U85 small CB-specific RNA, U2 small nuclear RNA, the survival of motor neurons protein, and fibrillarin occur together in a nuclear body that is closely associated with the nucleolus. Based on its similarity to CBs in other organisms, we refer to this structure as the D. melanogaster CB. Surprisingly, the D. melanogaster U7 small nuclear RNP resides in a separate nuclear body, which we call the histone locus body (HLB). The HLB is invariably colocalized with the histone gene locus. Thus, canonical CB components are distributed into at least two nuclear bodies in D. melanogaster. The identification of these nuclear bodies now permits a broad range of questions to be asked about CB structure and function in a genetically tractable organism.
Assuntos
Núcleo Celular/genética , Corpos Enovelados/genética , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , RNA Mensageiro/genética , Ribonucleoproteína Nuclear Pequena U7/genética , Animais , Animais Geneticamente Modificados , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Corpos Enovelados/metabolismo , Corpos Enovelados/ultraestrutura , Drosophila melanogaster/citologia , Embrião não Mamífero/citologia , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Evolução Molecular , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Histonas/genética , Histonas/metabolismo , Histonas/ultraestrutura , Larva/citologia , Larva/crescimento & desenvolvimento , Larva/metabolismo , Família Multigênica/fisiologia , Biossíntese de Proteínas/fisiologia , RNA Mensageiro/metabolismo , Ribonucleoproteína Nuclear Pequena U7/metabolismo , Ribonucleoproteína Nuclear Pequena U7/ultraestrutura , Ribonucleoproteínas Nucleares Pequenas/genética , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Especificidade da EspécieRESUMO
All retroviruses must circumvent cellular restrictions on the export of unspliced RNAs from the nucleus. While the unspliced RNA export pathways for HIV and Mason-Pfizer monkey virus are well characterized, that of Rous sarcoma virus (RSV) is not. We have previously reported that the RSV direct repeat (DR) elements are involved in the cytoplasmic accumulation of unspliced viral RNA. Here, using fluorescent in situ hybridization (FISH), we demonstrate that unspliced viral RNAs bearing a single point mutation (G8863C) in the DR exhibit a restricted cellular localization in and around the nucleus. In contrast, wild type unspliced viral RNA had a diffuse localization throughout the nucleus and cytoplasm. Since the RSV Gag protein has a transient localization in the nucleus, we examined the effect of Gag over-expression on a DR-mediated reporter construct. While Gag did not enhance DR-mediated nuclear export, the dominant-negative expression of two cellular export factors, Tap and Dbp5, inhibited expression of the same reporter construct. Furthermore, FISH studies using the dominant-negative Dbp5 demonstrated that unspliced wild type RSV RNA was retained within the nucleus. Taken together, these results further implicate the DR in nuclear RNA export through interactions with Tap and Dbp5.