Meet the Authors: Fardin Aryan, Diego Detrés, and Eliezer Calo.

We talk to co-first authors Fardin Aryan and Diego Detrés along with lead contact Eliezer Calo about their paper "Nucleolus activity-dependent recruitment and biomolecular condensation by pH sensing" (this issue of Molecular Cell), what drew them to a career in science, and overcoming challenges posed by the global pandemic.

Nucleolus activity-dependent recruitment and biomolecular condensation by pH sensing.

DEAD-box ATPases are major regulators of biomolecular condensates and orchestrate diverse biochemical processes that are critical for the functioning of cells. How DEAD-box proteins are selectively recruited to their respective biomolecular condensates is unknown. We explored this in the context of the nucleolus and DEAD-box protein DDX21. We find that the pH of the nucleolus is intricately linked to the transcriptional activity of the organelle and facilitates the recruitment and condensation of DDX21. We identify an evolutionarily conserved feature of the C terminus of DDX21 responsible for nucleolar localization. This domain is essential for zebrafish development, and its intrinsically disordered and isoelectric properties are necessary and sufficient for the ability of DDX21 to respond to changes in pH and form condensates. Molecularly, the enzymatic activities of poly(ADP-ribose) polymerases contribute to maintaining the nucleolar pH and, consequently, DDX21 recruitment and nucleolar partitioning. These observations reveal an activity-dependent physicochemical mechanism for the selective recruitment of biochemical activities to biomolecular condensates.

DNA-PKcs has KU-dependent function in rRNA processing and haematopoiesis.

The DNA-dependent protein kinase (DNA-PK), which comprises the KU heterodimer and a catalytic subunit (DNA-PKcs), is a classical non-homologous end-joining (cNHEJ) factor1. KU binds to DNA ends, initiates cNHEJ, and recruits and activates DNA-PKcs. KU also binds to RNA, but the relevance of this interaction in mammals is unclear. Here we use mouse models to show that DNA-PK has an unexpected role in the biogenesis of ribosomal RNA (rRNA) and in haematopoiesis. The expression of kinase-dead DNA-PKcs abrogates cNHEJ2. However, most mice that both expressed kinase-dead DNA-PKcs and lacked the tumour suppressor TP53 developed myeloid disease, whereas all other previously characterized mice deficient in both cNHEJ and TP53 expression succumbed to pro-B cell lymphoma3. DNA-PK autophosphorylates DNA-PKcs, which is its best characterized substrate. Blocking the phosphorylation of DNA-PKcs at the T2609 cluster, but not the S2056 cluster, led to KU-dependent defects in 18S rRNA processing, compromised global protein synthesis in haematopoietic cells and caused bone marrow failure in mice. KU drives the assembly of DNA-PKcs on a wide range of cellular RNAs, including the U3 small nucleolar RNA, which is essential for processing of 18S rRNA4. U3 activates purified DNA-PK and triggers phosphorylation of DNA-PKcs at T2609. DNA-PK, but not other cNHEJ factors, resides in nucleoli in an rRNA-dependent manner and is co-purified with the small subunit processome. Together our data show that DNA-PK has RNA-dependent, cNHEJ-independent functions during ribosome biogenesis that require the kinase activity of DNA-PKcs and its phosphorylation at the T2609 cluster.

Tissue-selective effects of nucleolar stress and rDNA damage in developmental disorders.

Many craniofacial disorders are caused by heterozygous mutations in general regulators of housekeeping cellular functions such as transcription or ribosome biogenesis. Although it is understood that many of these malformations are a consequence of defects in cranial neural crest cells, a cell type that gives rise to most of the facial structures during embryogenesis, the mechanism underlying cell-type selectivity of these defects remains largely unknown. By exploring molecular functions of DDX21, a DEAD-box RNA helicase involved in control of both RNA polymerase (Pol) I- and II-dependent transcriptional arms of ribosome biogenesis, we uncovered a previously unappreciated mechanism linking nucleolar dysfunction, ribosomal DNA (rDNA) damage, and craniofacial malformations. Here we demonstrate that genetic perturbations associated with Treacher Collins syndrome, a craniofacial disorder caused by heterozygous mutations in components of the Pol I transcriptional machinery or its cofactor TCOF1 (ref. 1), lead to relocalization of DDX21 from the nucleolus to the nucleoplasm, its loss from the chromatin targets, as well as inhibition of rRNA processing and downregulation of ribosomal protein gene transcription. These effects are cell-type-selective, cell-autonomous, and involve activation of p53 tumour-suppressor protein. We further show that cranial neural crest cells are sensitized to p53-mediated apoptosis, but blocking DDX21 loss from the nucleolus and chromatin rescues both the susceptibility to apoptosis and the craniofacial phenotypes associated with Treacher Collins syndrome. This mechanism is not restricted to cranial neural crest cells, as blood formation is also hypersensitive to loss of DDX21 functions. Accordingly, ribosomal gene perturbations associated with Diamond-Blackfan anaemia disrupt DDX21 localization. At the molecular level, we demonstrate that impaired rRNA synthesis elicits a DNA damage response, and that rDNA damage results in tissue-selective and dosage-dependent effects on craniofacial development. Taken together, our findings illustrate how disruption in general regulators that compromise nucleolar homeostasis can result in tissue-selective malformations.

An evolutionarily nascent architecture underlying the formation and emergence of biomolecular condensates.

Biomolecular condensates are implicated in core cellular processes such as gene regulation and ribosome biogenesis. Although the architecture of biomolecular condensates is thought to rely on collective interactions between many components, it is unclear how the collective interactions required for their formation emerge during evolution. Here, we show that the structure and evolution of a recently emerged biomolecular condensate, the nucleolar fibrillar center (FC), is explained by a single self-assembling scaffold, TCOF1. TCOF1 is necessary to form the FC, and it structurally defines the FC through self-assembly mediated by homotypic interactions of serine/glutamate-rich low-complexity regions (LCRs). Finally, introduction of TCOF1 into a species lacking the FC is sufficient to form an FC-like biomolecular condensate. By demonstrating that a recently emerged biomolecular condensate is built on a simple architecture determined by a single self-assembling protein, our work provides a compelling mechanism by which biomolecular condensates can emerge in the tree of life.