RESUMEN
Drosophila PCID2 is a subunit of the TREX-2 mRNA nuclear export complex. Although the complex has long been studied in eukaryotes, it is still unclear how TREX-2 interacts with mRNA in multicellular organisms. Here, the interaction between Drosophila PCID2 and the ras2 RNA was studied by EMSA. We show that the C-terminal region of the WH domain of PCID2 specifically binds the 3'-noncoding region of the ras2 RNA. While the same region of PCID2 interacts with the Xmas-2 subunit of the TREX-2 complex, PCID2 interacts with RNA independently of Xmas-2. An additional RNA-binding region (M region) was identified in the N-terminal part of the PCI domain and found to bind RNA nonspecifically. Point mutations of evolutionarily conserved amino acid residues in this region completely abolish the PCID2-RNA interaction, while a deletion of the C-terminal domain only partly decreases it. Thus, the specific interaction of PCID2 with RNA requires nonspecific PCID2-RNA binding.
RESUMEN
Understanding the functions of TBP-related factors is essential for studying chromatin assembly and transcription regulation in higher eukaryotes. The novel TBP-related protein-coding gene, trf4, was described in Drosophila melanogaster. trf4 is found only in Drosophila and has likely originated in Drosophila common ancestor. TRF4 protein has a distant homology with TBP and TRF2 in the region of TBP-like domain and is evolutionarily conserved among distinct Drosophila species, which indicates its functional significance. TRF4 is widely expressed in D. melanogaster with high levels of its expression being observed in testes. Interestingly enough, TRF4 has become a cytoplasmic protein having lost nuclear localization signal sequence. TRF4 is concentrated at the endoplasmic reticulum (ER) and copurifies with the proteins participating in the ER-associated processes. We suggest that trf4 gene is an example of homolog neofunctionalization by protein subcellular relocalization pathway, where the subcellular relocalization of gene product of duplicated gene leads to the new functions in ER-associated processes.
RESUMEN
SAGA/TFTC-type multiprotein complexes play important roles in the regulation of transcription. We have investigated the importance of the nuclear positioning of a gene, its transcription and the consequent export of the nascent mRNA. We show that E(y)2 is a subunit of the SAGA/TFTC-type histone acetyl transferase complex in Drosophila and that E(y)2 concentrates at the nuclear periphery. We demonstrate an interaction between E(y)2 and the nuclear pore complex (NPC) and show that SAGA/TFTC also contacts the NPC at the nuclear periphery. E(y)2 forms also a complex with X-linked male sterile 2 (Xmas-2) to regulate mRNA transport both in normal conditions and after heat shock. Importantly, E(y)2 and Xmas-2 knockdown decreases the contact between the heat-shock protein 70 (hsp70) gene loci and the nuclear envelope before and after activation and interferes with transcription. Thus, E(y)2 and Xmas-2 together with SAGA/TFTC function in the anchoring of a subset of transcription sites to the NPCs to achieve efficient transcription and mRNA export.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Complejos Multiproteicos/metabolismo , Poro Nuclear/metabolismo , Transporte de ARN/fisiología , ARN Mensajero/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética , Animales , Línea Celular , Núcleo Celular/metabolismo , Cromosomas/metabolismo , Microscopía por Crioelectrón , Proteínas de Drosophila/genética , Proteínas de Drosophila/ultraestructura , Drosophila melanogaster/genética , Regulación de la Expresión Génica , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Humanos , Inmunohistoquímica , Masculino , Complejos Multiproteicos/química , Membrana Nuclear/metabolismo , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Interferencia de ARN , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/ultraestructura , Técnicas del Sistema de Dos HíbridosRESUMEN
The Drosophila TATA box-binding protein (TBP)-related factor 2 (TRF2 or TLF) was shown to control a subset of genes different from that controlled by TBP. Here, we have investigated the structure and functions of the trf2 gene. We demonstrate that it encodes two protein isoforms: the previously described 75-kDa TRF2 and a newly identified 175-kDa version in which the same sequence is preceded by a long N-terminal domain with coiled-coil motifs. Chromatography of Drosophila embryo extracts revealed that the long TRF2 is part of a multiprotein complex also containing ISWI. Both TRF2 forms are detected at the same sites on polytene chromosomes and have the same expression patterns, suggesting that they fulfill similar functions. A study of the manifestations of the trf2 mutation suggests an essential role of TRF2 during embryonic Drosophila development. The trf2 gene is strongly expressed in germ line cells of adult flies. High levels of TRF2 are found in nuclei of primary spermatocytes and trophocytes with intense transcription. In ovaries, TRF2 is present both in actively transcribing nurse cells and in the transcriptionally inactive oocyte nuclei. Moreover, TRF2 is essential for premeiotic chromatin condensation and proper differentiation of germ cells of both sexes.
Asunto(s)
Diferenciación Celular , Cromatina/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriología , Drosophila melanogaster/metabolismo , Células Germinativas/citología , Meiosis , Proteína 2 de Unión a Repeticiones Teloméricas/metabolismo , Aminoácidos/genética , Aminoácidos/metabolismo , Animales , Proteínas de Drosophila/genética , Drosophila melanogaster/citología , Drosophila melanogaster/genética , Femenino , Regulación del Desarrollo de la Expresión Génica , Células Germinativas/metabolismo , Masculino , Peso Molecular , Mutación/genética , Sistemas de Lectura Abierta/genética , Unión Proteica , Biosíntesis de Proteínas/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/genética , Espermatogénesis , Proteína 2 de Unión a Repeticiones Teloméricas/genética , Transcripción Genética/genéticaRESUMEN
The e(y)2 gene of Drosophila melanogaster encodes the ubiquitous evolutionarily conserved co-activator of RNA polymerase II that is involved in transcription regulation of a high number of genes. The Drosophila e(y)2b gene, paralogue of the e(y)2 has been found. The analysis of structure of the e(y)2, e(y)2b and its orthologues from other species reveals that the e(y)2 gene derived as a result of retroposition of the e(y)2b during Drosophila evolution. The mRNA-derived retrogenes lack introns or regulatory regions; most of them become pseudogenes whereas some acquire tissue-specific functions. Here we describe the different situation: the e(y)2 retrogene performs the general function and is ubiquitously expressed, while the source gene is functional only in a small group of male germ cells. This must have resulted from retroposition into a transcriptionally favorable region of the genome.