ABSTRACT

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.

摘要

从核仁内部区域获得完整的前核糖体复合物的技术困难阻碍了人类核糖体合成途径的生化研究。在这里，我们提供了一种提取方法的详细描述，该方法能够有效地检测、分离和表征含有大型 pre-rRNA 种类的核仁前核糖体。三步前核糖体序列提取 (PSE) 协议保留了早期前核糖体复合物的完整性，并产生适合于从少量起始材料进行生化分析的制剂。我们通过使用亲和矩阵下拉和沉降分析检测提取的前核糖体中的特定反式作用因子和前 rRNA 来验证这一过程。此外，我们描述了 PSE 方法在监测无核糖体 5S RNP 复合物的细胞水平中的应用，作为核糖体生物发生压力的指标。我们优化的实验程序将有助于研究正常、突变和应激细胞情景中的人类核糖体生物发生，包括表征候选核糖体生物发生因子、特定生理条件下的前核糖体相互作用物或药物对核糖体成熟的影响。