Proteomic profiling of early pre-ribosomal complexes from human nucleolar extracts

The nucleolus is a key cellular compartment responsible for the major steps of ribosomal subunit biogenesis. Detailed characterization of the nucleolar proteome is essential for understanding the molecular mechanisms underlying ribosome biogenesis and may help identify novel regulators of this process. However, the nucleolar proteome, and the protein composition of early ribosomal subunit precursors in particular, remain poorly defined, largely because of the methodological inconsistencies and conflicting findings across published studies. Here, we aimed to establish a standardized approach that could be used to identify novel factors involved in the early stages of ribosome biogenesis using an optimized stepwise nucleolar extraction protocol, followed by sucrose density gradient fractionation and mass spectrometry analysis. This optimized approach allowed us to enrich for proteins directly engaged in early biogenesis steps, while depleting non-specific nucleolar “background” components. A total of 529 unique proteins were identified across two biological replicates. Gene Ontology (GO) analysis revealed significant enrichment of proteins annotated as ribosome biogenesis factors, including those directly involved in ribosomal subunit assembly and rRNA processing. A cross-referencing of our dataset against 188 known components of pre-ribosomal complexes represented in the PDB yielded 84 overlapping proteins. Early biogenesis factors prevailed in this group (70 out of 84), supporting the idea of the enrichment of the analyzed fraction with early pre-ribosomal complexes. The resulting protein dataset will facilitate the identification of novel factors governing the poorly understood aspects of human ribosome biogenesis.