Viscoelastic RNA entanglement and advective flow underlie nucleolar form and function

The nucleolus facilitates transcription, processing, and assembly of ribosomal RNA (rRNA), the most abundant RNA in cells. Nucleolar function is facilitated by its multiphase liquid properties, but nucleolar fluidity and its connection to ribosome biogenesis remain unclear. Here, we used quantitative imaging, mathematical modeling, and pulse-chase nucleotide labelling to map nucleolar rRNA dynamics. Inconsistent with a purely diffusive process, rRNA steadily expands away from the transcriptional sites, moving in a slow (~1 Å/s), radially-directed fashion. This motion reflects the viscoelastic properties of a highly concentrated gel of entangled rRNA, whose constant polymerization drives steady outward flow. We propose a new viscoelastic rRNA release model, where nucleolar rRNA cleavage and processing reduce entanglement, fluidizing the nucleolar periphery to facilitate release of mature pre-ribosomal particles.

A high-throughput assay for directly monitoring nucleolar rRNA biogenesis

Studies of the regulation of nucleolar function are critical for ascertaining clearer insights into the basic biological underpinnings of ribosome biogenesis, and for future development of therapeutics to treat cancer and ribosomopathies. A number of high-throughput primary assays based on morphological alterations of the nucleolus can indirectly identify hits affecting ribosome biogenesis. However, there is a need for a more direct high-throughput assay for nucleolar function to further evaluate hits. Previous reports have monitored nucleolar RNA biogenesis using 5-ethynyl uridine (5-EU) in low-throughput. We report a miniaturized, high-throughput 5-EU assay for nucleolar function which enables specific calculation of nucleolar rRNA biogenesis inhibition, based on co-staining of the nucleolar protein fibrillarin (FBL). The assay utilizes two siRNA controls, a negative non-targeting siRNA control (siNT) and a positive siRNA control targeting POLR1A (siPOLR1A), and specifically quantifies median 5-EU signal within nucleoli. Maximum nuclear 5-EU signal can also be used to monitor the effects of putative small molecule inhibitors of RNAP1, like BMH-21, or other treatment conditions that cause FBL dissociation. We validate the 5-EU assay on 68 predominately nucleolar hits from a high-throughput primary screen, showing that 58/68 hits significantly inhibit nucleolar rRNA biogenesis. Our new method establishes direct quantification of nucleolar function in high-throughput, facilitating closer study of ribosome biogenesis in health and disease.

The cohesin regulator Stag1 promotes cell plasticity through heterochromatin regulation

SUMMARYFundamental biological processes such as embryo development and stem cell control rely on cellular plasticity. We present a role for the cohesin regulator, Stag1 in cellular plasticity control via heterochromatin regulation. Stag1 localises to heterochromatin domains and repetitive sequences in embryonic stem (ES) cells and contains intrinsically disordered regions in its divergent terminal ends which promote heterochromatin compaction. ES cells express Stag1 protein isoforms lacking the disordered ends and fluctuations in isoform abundance skews the cell state continuum towards increased differentiation or reprogramming. The role for Stag1 in heterochromatin condensates and nucleolar function is dependent on its unique N-terminus. Stag1NΔ ESCs have decompacted chromatin and reprogram towards totipotency, exhibiting MERVL derepression, reduced nucleolar transcription and decreased translation. Our results move beyond protein-coding gene regulation via chromatin loops into a new role for Stag1 in heterochromatin and nucleolar function and offer fresh perspectives on its contribution to cell identity and disease.

The Ribosome Biogenesis—Cancer Connection

Multifaceted relations link ribosome biogenesis to cancer. Ribosome biogenesis takes place in the nucleolus. Clarifying the mechanisms involved in this nucleolar function and its relationship with cell proliferation: 1) allowed the understanding of the reasons for the nucleolar changes in cancer cells and their exploitation in tumor pathology, 2) defined the importance of the inhibition of ribosome biogenesis in cancer chemotherapy and 3) focused the attention on alterations of ribosome biogenesis in the pathogenesis of cancer. This review summarizes the research milestones regarding these relevant relationships between ribosome biogenesis and cancer. The structure and function of the nucleolus will also be briefly described.

Selective degeneration of dopamine neurons in Parkinson’s disease: emerging roles of altered calcium homeostasis and nucleolar function

Parkinson’s disease (PD) is the second most common neurodegenerative disease. Its classic major motor-symptoms are caused by the progressive loss of dopamine in the striatum, and of dopamine (DA) releasing neurons in the midbrain, particularly within the Substantia nigra (SN). The cause for PD is still unclear, hampering the development of curative therapies. However multiple genetic and environmental PD trigger factors have been identified, pointing to a common, mutually interdependent pathomechanism of cell-specific metabolic dysfunction and altered gene expression. Here, we summarize and discuss these emerging PD-pathomechanisms, that could provide novel potential therapeutic targets, with a focus on altered Ca2+ homeostasis and nucleolar function. We discuss how animal models with impaired nucleolar ribosomal RNA synthesis can enable identification of novel cell-specific vulnerability factors, and how complex homeostatic adaptation of SN DA neurons could enable a flexible adjustment of their functional activity to metabolic needs, but also might render them particularly vulnerable to degenerative triggers and cell-death in PD.