Nucleolar Organization in Response to Transcriptional Stress

Abstract

The nucleolus, a prominent membrane-less nuclear compartment, is organized around ribosomal RNA (rRNA) gene (rDNA) clusters, known as nucleolar organizing regions (NORs), located on the short arms of acrocentric chromosomes. It serves as the primary site for ribosome biogenesis, an energy-intensive process crucial for cell growth and proliferation. This involves RNA polymerase I (Pol I)-mediated transcription of 47S precursor rRNA (pre-rRNA), pre-rRNA processing, and ribosomal subunit assembly, reflected in its tripartite structure maintained by liquid-liquid phase separation. Recent evidence indicates that only about 30% of nucleolar proteins are exclusively involved in ribosome production. The remaining proteome participates in diverse cellular functions, establishing the nucleolus as a multifunctional organelle. It functions as a critical stress sensor and signaling hub, responding to various intracellular insults such as nutrient starvation, DNA damage, and viral infection. Many chemotherapeutic agents also induce the response called nucleolar stress via disruption of the nucleolar structure or function, potentially leading to rDNA instability. Nucleolar stress frequently leads to dynamic transition of nucleolar proteins, inducing nucleolar reorganization. Of these, the stress induced by transcriptional changes leads to the unique nucleolar structures termed nucleolar caps and nucleolar necklaces. In this review, we summarize the recent findings about the molecular mechanism of nucleolar changes upon stresses and discuss the possible relationship between rDNA instability and cancer.

Keywords: cancer; liquid–liquid phase separation; nucleolus; rDNA stability; transcriptional stress.

FIGURE 1

Schematic representation of ribosomal RNA (rRNA) genes in human. The rRNA gene array is located at the nucleolar organizer regions (NORs). It is positioned between telomeres and centromeres on the short arms (p‐arms) of the acrocentric chromosomes 13, 14, 15, 21, and 22 in humans. They are flanked by heterochromatic distal and proximal junctions (DJ and PJ, respectively). Each ribosomal DNA (rDNA) repeat unit consists of an intergenic spacer (IGS) and a transcribed region encoding the 47S pre‐rRNA, which is further processed into the mature 18S, 5.8S, and 28S rRNAs. The coding region is flanked by external transcribed spacers (5′ ETS and 3′ ETS), and rDNA genes are separated by internal transcribed spacers (ITS1 and ITS2). The IGS includes the promoter for rDNA transcription.

FIGURE 2

Nucleolar organization and its function. The overall structure of the mammalian nucleolus consists of the internal fibrillar center (FC), dense fibrillar component (DFC), and granular component (GC), all of which are surrounded by condensed perinucleolar heterochromatin (PH). RNA polymerase I (Pol I) transcribes the rRNA genes to form 47S pre‐rRNA at the FC‐DFC border. The transcription factor upstream binding factor (UBF) and Treacle (TCOF1) are required for rDNA transcription. In the DFC, pre‐rRNA is further processed and modified to produce the mature 18S, 5.8S, and 28S rRNAs. The DFC contains small nucleolar ribonucleoproteins (snoRNPs), Fibrillarin (FBL), and Nucleolin (NCL). In the GC, pre‐40S and pre‐60S are assembled and exported to the nucleoplasm. The GC harbors nucleophosmin (NPM1).

FIGURE 3

Nucleolar reorganization upon transcriptional stress. Upon Pol I inhibition, rDNA together with proteins of the FC and DFC sub‐compartments moves to the nucleolar periphery, forming nucleolar caps. Upon Pol II inhibition, the GC is disrupted and the FC and DFC modules are unraveled, forming CITIs and/or nucleolar necklaces.

FIGURE 4

The nucleolar DNA damage response (n‐DDR). In the left panel, upon the presence of persistent rDNA double‐strand breaks (DSBs) in the nucleolus, the ataxia telangiectasia mutated (ATM) kinase is activated and phosphorylates TCOF1. The MRN complex, consisting of Nijmegen breakage syndrome protein 1 (NBS1), meiotic recombination 11 homolog 1 (MRE11), and the DNA repair protein RAD50, is recruited to the nucleolus through the interaction between NBS1 and phospho‐TCOF1. Phosphorylated TCOF1 recruits DNA topoisomerase II binding protein 1 (TOPBP1), activating the ataxia telangiectasia and Rad3‐related protein (ATR) kinase to elicit transcription silencing. UFL1 involves transcription silencing via TCOF1 modifications. In the right panel, transcription silencing elicits the nucleolar cap formation. The nucleolar caps include the homologous recombination (HR) factors: Breast cancer gene 1 (BRCA1), replication protein A2 (RPA2), carboxy‐terminal binding protein interacting protein (CtIP), and radiation sensitive protein 51 (RAD51). ATM, ATR, the MRN complex, the phosphorylated form of the histone variant H2AX (γH2AX), and p53‐binding protein 1 (53BP1) are clustered at nucleolar caps.