Efficient fractionation and analysis of ribosome assembly intermediates in human cells

© 2021 The Author(s).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.This work was supported by the Spanish Ministry of Science and Innovation [BFU2017-88192-P to MD][RTI2018-096481-B-100 to XRB]; the National Institutes of Health [R03CA246009 to DGP]; the Castilla-León autonomous government [CSI252P18 and CSI145P2 to XRB]; the Spanish Association against Cancer [GC16173472GARC to XRB]; “la Caixa” Banking Foundation [HR20-00164 to XRB]; and the Samuel Solórzano Foundation [FS/36-2017 to MD]. BN has been supported by a predoctoral contract sponsored by the University of Salamanca and Santander Bank, SGG by a predoctoral FPU contract of the Spanish Ministry of Education, Culture and Sports, and LC by a predoctoral contract from the Spanish Association against Cancer. The Centro de Investigación del Cáncer is supported by the Programa de Apoyo a Planes Estratégicos de Investigación de Estructuras de Investigación de Excelencia of the Ministry of Education of the Castilla-León Government (CLC-2017-01). Both the Spanish and Castilla-León government-associated funding is partially supported by the European Regional Development Fund