Efficient fractionation and analysis of ribosome assembly intermediates in human cells
[EN]Biochemical studies of the human ribosome synthesis pathway have been hindered by technical difficulties in obtaining intact preribosomal complexes from internal regions of the nucleolus. Here we provide a detailed description of an extraction method that enables efficient detection, isolation,...
| Autores: | , , , , , , |
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| Tipo de recurso: | artículo |
| Estado: | Versión publicada |
| Fecha de publicación: | 2021 |
| País: | España |
| Institución: | Universidad de Salamanca (USAL) |
| Repositorio: | GREDOS. Repositorio Institucional de la Universidad de Salamanca |
| OAI Identifier: | oai:gredos.usal.es:10366/168975 |
| Acceso en línea: | http://hdl.handle.net/10366/168975 |
| Access Level: | acceso abierto |
| Palabra clave: | Preribosome Human nucleolus pre-rRNA ribosome maturation ribosome assembly preribosome purification ribosome production RNA Ribosomal Proteins Ribosomes Humans Cell Nucleolus HCT116 Cells HeLa Cells RNA Precursors 2415 Biología Molecular 2302 Bioquímica precursores del ARN nucleolo celular humanos ARN células HCT116 células HeLa proteínas ribosómicas ribosomas |
| Sumario: | [EN]Biochemical studies of the human ribosome synthesis pathway have been hindered by technical difficulties in obtaining intact preribosomal complexes from internal regions of the nucleolus. Here we provide a detailed description of an extraction method that enables efficient detection, isolation, and characterization of nucleolar preribosomes containing large pre-rRNA species. The three-step Preribosome Sequential Extraction (PSE) protocol preserves the integrity of early preribosomal complexes and yields preparations amenable to biochemical analyses from low amounts of starting material. We validate this procedure through the detection of specific trans-acting factors and pre-rRNAs in the extracted preribosomes using affinity matrix pull-downs and sedimentation assays. In addition, we describe the application of the PSE method for monitoring cellular levels of ribosome-free 5S RNP complexes as an indicator of ribosome biogenesis stress. Our optimized experimental procedures will facilitate studies of human ribosome biogenesis in normal, mutant and stressed-cell scenarios, including the characterization of candidate ribosome biogenesis factors, preribosome interactors under specific physiological conditions or effects of drugs on ribosome maturation. |
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