Efficient fractionation and analysis of ribosome assembly intermediates in human cells

RNA Biol. 2021 Oct 15;18(sup1):182-197. doi: 10.1080/15476286.2021.1965754. Epub 2021 Sep 17.


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.

Keywords: Human ribosome synthesis; pre-rRNA maturation; preribosomes; ribosome assembly.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Nucleolus / genetics
  • Cell Nucleolus / metabolism*
  • HCT116 Cells
  • HeLa Cells
  • Humans
  • RNA Precursors / genetics
  • RNA Precursors / metabolism*
  • RNA, Ribosomal / genetics
  • RNA, Ribosomal / metabolism*
  • Ribosomal Proteins / genetics
  • Ribosomal Proteins / isolation & purification*
  • Ribosomal Proteins / metabolism*
  • Ribosomes / genetics
  • Ribosomes / metabolism*


  • RNA Precursors
  • RNA, Ribosomal
  • Ribosomal Proteins