Among numerous microscopically visible nuclear substructures, the nucleolus is the most prominent and represents a functionally and biophysically distinct body or compartment. The nucleolus is, in fact, so prominent that it drew the attention of early biologists over 200 years ago, in light microscopy studies by Fontana, Valentin and Wagner 1. Following these early descriptions came the understanding of the functional importance of the nucleolus, including its primary role as the site for the initial steps of ribosome biogenesis, including RNA polymerase I (Pol I)-driven transcription, processing and modification of ribosomal RNA (rRNA) and the assembly of rRNA-containing complexes 2 (Fig. 1). These processes involve several hundred protein transacting factors and small nucleolar RNAs 3 , which serve to guide the specificity of rRNA chemical modifications, pre-rRNA folding and cleavage. Once precursor subunits are released from the nucleolar structure, they undergo further maturation in the nucleoplasm and cytoplasm prior to becoming fully functional ribosomal subunits, ready to engage in translating mRNA into protein. This nucleolar function is accompanied by organization of the nucleolus into distinct subcompartments. In mammalian cells, nucleoli display three layers, nested like Russian dolls, where successive steps of ribosome production take place, starting

ABSTRACTrRNAs are extensively modified during their transcription and subsequent maturation in the nucleolus, nucleus and cytoplasm. RNA modifications, which are installed either by snoRNA-guided or by stand-alone enzymes, generally stabilize the structure of the ribosome. However, they also cluster at functionally important sites of the ribosome, such as the peptidyltransferase center and the decoding site, where they facilitate efficient and accurate protein synthesis. The recent identification of sites of substoichiometric 2′-O-methylation and pseudouridylation has overturned the notion that all rRNA modifications are constitutively present on ribosomes, highlighting nucleotide modifications as an important source of ribosomal heterogeneity. While the mechanisms regulating partial modification and the functions of specialized ribosomes are largely unknown, changes in the rRNA modification pattern have been observed in response to environmental changes, during development, and in disease. This suggests that rRNA modifications may contribute to the translational control of gene expression.

Mature ribosomal RNAs (rRNAs) are produced from polycistronic precursors following complex processing. Precursor (pre)-rRNA processing has been extensively characterized in yeast and was assumed to be conserved in humans. We functionally characterized 625 nucleolar proteins in HeLa cells and identified 286 required for processing, including 74 without a yeast homolog. For selected candidates, we demonstrated that pre-rRNA processing defects are conserved in different cell types (including primary cells), defects are not due to activation of a p53-dependent nucleolar tumor surveillance pathway, and they precede cell-cycle arrest and apoptosis. We also investigated the exosome's role in processing internal transcribed spacers (ITSs) and report that 3' end maturation of 18S rRNA involves EXOSC10/Rrp6, a yeast ITS2 processing factor. We conclude that human cells adopt unique strategies and recruit distinct trans-acting factors to carry out essential processing steps, posing fundamental implications for understanding ribosomopathies at the molecular level and developing effective therapeutic agents.