RESUMO
Ribosome production, one of the most energy-consuming biosynthetic activities in living cells, is adjusted to growth conditions and coordinated with the cell cycle. Connections between ribosome synthesis and cell cycle progression have been described, but the underlying mechanisms remain only partially understood. The human HCA66 protein was recently characterized as a component of the centrosome, the major microtubule-organizing center (MTOC) in mammalian cells, and was shown to be required for centriole duplication and assembly of the mitotic spindle. We show here that HCA66 is also required for nucleolar steps of the maturation of the 40S ribosomal subunit and therefore displays a dual function. Overexpression of a dominant negative version of HCA66, accumulating at the centrosome but absent from the nucleoli, alters centrosome function but has no effect on pre-rRNA processing, suggesting that HCA66 acts independently in each process. In yeast and HeLa cells, depletion of MTOC components does not impair ribosome synthesis. Hence our results suggest that both in yeast and human cells, assembly of a functional MTOC and ribosome synthesis are not closely connected processes.
Assuntos
Antígenos de Neoplasias/metabolismo , Proteínas de Transporte/metabolismo , Centríolos/fisiologia , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Nucléolo Celular/metabolismo , Centrossomo/metabolismo , Células HeLa , Humanos , Proteínas Nucleares/metabolismo , Precursores de RNA/metabolismo , RNA Ribossômico/metabolismo , RNA Ribossômico 18S/metabolismo , Proteínas de Ligação a RNA , Ribossomos/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/metabolismoRESUMO
Ribosome biogenesis in eukaryotes is a major cellular activity mobilizing the products of over 200 transcriptionally coregulated genes referred to as the rRNA and ribosome biosynthesis regulon. We investigated the function of an essential, uncharacterized gene of this regulon, renamed RRP36. We show that the Rrp36p protein is nucleolar and interacts with 90S and pre-40S preribosomal particles. Its depletion affects early cleavages of the 35S pre-rRNA and results in a rapid decrease in mature 18S rRNA levels. Rrp36p is a novel component of the 90S preribosome, the assembly of which has been suggested to result from the stepwise incorporation of several modules, including the tUTP/UTP-A, PWP2/UTP-B, and UTP-C subcomplexes. We show that Rrp36p depletion does not impair the incorporation of these subcomplexes and the U3 small nucleolar RNP into preribosomes. In contrast, depletion of components of the UTP-A or UTP-B modules, but not Rrp5p, prevents Rrp36p recruitment and reduces its accumulation levels. In parallel, we studied the human orthologue of Rrp36p in HeLa cells, and we show that the function of this protein in early cleavages of the pre-rRNA has been conserved through evolution in eukaryotes.
Assuntos
Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , RNA Fúngico/genética , RNA Fúngico/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/genética , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Nucléolo Celular/metabolismo , Sequência Conservada , Evolução Molecular , Genes Fúngicos , Células HeLa , Humanos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Interferência de RNA , RNA Interferente Pequeno/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Regulon , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , TransfecçãoRESUMO
BACKGROUND: The publication of the first draft chicken sequence assembly became available in 2004 and was updated in 2006. However, this does not constitute a definitive and complete sequence of the chicken genome, since the microchromosomes are notably under-represented. In an effort to develop maps for the microchromosomes absent from the chicken genome assembly, we developed radiation hybrid (RH) and genetic maps with markers isolated from sequence currently assigned to "chromosome Unknown" (chrUn). The chrUn is composed of sequence contigs not assigned to named chromosomes. To identify and map sequence belonging to the microchromosomes we used a comparative mapping strategy, and we focused on the small linkage group E26C13. RESULTS: In total, 139 markers were analysed with the chickRH6 panel, of which 120 were effectively assigned to the E26C13 linkage group, the remainder mapping elsewhere in the genome. The final RH map is composed of 22 framework markers extending over a 245.6 cR distance. A corresponding genetic map was developed, whose length is 103 cM in the East Lansing reference population. The E26C13 group was assigned to GGA25 (Gallus gallus chromosome 25) by FISH (fluorescence in situ hybridisation) mapping. CONCLUSION: The high-resolution RH framework map obtained here covers the entire chicken chromosome 25 and reveals the existence of a high number of intrachromosomal rearrangements when compared to the human genome. The strategy used here for the characterization of GGA25 could be used to improve knowledge on the other uncharacterized small, yet gene-rich microchromosomes.
