Abstract
The eukaryotic nucleolus is a highly organized, multilayered structure essential for ribosomal RNA (rRNA) processing and ribosome assembly. While rRNA transcription is known to drive nucleolar assembly, the contribution of downstream processing steps to nucleolar organization remains less well defined. Here, we show that disruption of endonucleolytic cleavage of the 5′ external transcribed spacer (5′ETS), a key early step mediated by the SSU processome, leads to pronounced changes in nucleolar structure and organization. These altered nucleoli display reduced dynamic exchange behavior consistent with altered material properties and exhibit changes in the NPM1-associated nucleolar interactome. In parallel, we observe redistribution of heterochromatin markers, indicating broader effects on nuclear organization. Together, our findings support a model in which progression of pre-rRNA processing contributes to maintaining nucleolar compartmentalization and links early steps of ribosome biogenesis to nuclear organization. The nucleolus is a defining feature of eukaryotic cells, yet the principles that maintain its multilayered organization remain incompletely understood. While rRNA transcription is known to initiate nucleolar assembly, we show that a specific downstream processing step, the cleavage of the 5′ external transcribed spacer (5′ETS), plays a key role in maintaining nucleolar organization. By functionally uncoupling rRNA transcription from processing, we demonstrate that defects in 5′ETS maturation are associated with alteration of the NPM1-associated interactome and changes in nucleolar dynamics. These findings support a model in which the progression of rRNA processing contributes to the material organization of the nucleolus, linking enzymatic steps in ribosome biogenesis to nuclear architecture.