Efficient fractionation and analysis of ribosome assembly intermediates in human cells.

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 descr

SUBMITTER: Nieto B 

PROVIDER: S-EPMC8682975 | biostudies-literature | 2021 Oct

REPOSITORIES: biostudies-literature