The mitotic checkpoint ensures accurate chromosome segregation through assembly of the mitotic checkpoint complex (MCC), a soluble inhibitor of the anaphase-promoting complex/cyclosome (APC/C) produced by unattached kinetochores. MCC is also assembled during interphase by Mad1/Mad2 bound at nuclear pores, thereby preventing premature mitotic exit prior to kinetochore maturation and checkpoint activation. Using degron tagging to rapidly deplete the AAA+ ATPase TRIP13, we show that its catalytic activity is required to maintain a pool of open-state Mad2 for MCC assembly, thereby supporting mitotic checkpoint activation, but is also required for timely mitotic exit through catalytic disassembly of MCC. Strikingly, combining TRIP13 depletion with elimination of APC15-dependent Cdc20 ubiquitination/degradation results in a complete inability to exit mitosis, even when MCC assembly at unattached kinetochores is prevented. Thus, mitotic exit requires MCC produced either in interphase or mitosis to be disassembled by TRIP13-catalyzed removal of Mad2 or APC15-driven ubiquitination/degradation of its Cdc20 subunit.

All living organisms have the ability to sense nutrient levels to coordinate cellular metabolism. Despite the importance of nutrient-sensing pathways that detect the levels of amino acids and glucose, how the availability of these two types of nutrients is integrated is unclear. Here, we show that glucose availability regulates the central nutrient effector mTORC1 through intracellular leucine sensor leucyl-tRNA synthetase 1 (LARS1). Glucose starvation results in O-GlcNAcylation of LARS1 on residue S1042. This modification inhibits the interaction of LARS1 with RagD GTPase and reduces the affinity of LARS1 for leucine by promoting phosphorylation of its leucine-binding site by the autophagy-activating kinase ULK1, decreasing mTORC1 activity. The lack of LARS1 O-GlcNAcylation constitutively activates mTORC1, supporting its ability to sense leucine, and deregulates protein synthesis and leucine catabolism under glucose starvation. This work demonstrates that LARS1 integrates leucine and glucose availability to regulate mTORC1 and the metabolic fate of leucine.

Haematopoietic stem and progenitor cells (HSPCs) have been the focus of developmental and regenerative studies, yet our understanding of the signalling events regulating their specification remains incomplete. We demonstrate that supt16h, a component of the Facilitates chromatin transcription (FACT) complex, is required for HSPC formation. Zebrafish supt16h mutants express reduced levels of Notch-signalling components, genes essential for HSPC development, due to abrogated transcription. Whereas global chromatin accessibility in supt16h mutants is not substantially altered, we observe a specific increase in p53 accessibility, causing an accumulation of p53. We further demonstrate that p53 influences expression of the Polycomb-group protein PHC1, which functions as a transcriptional repressor of Notch genes. Suppression of phc1 or its upstream regulator, p53, rescues the loss of both Notch and HSPC phenotypes in supt16h mutants. Our results highlight a relationship between supt16h, p53 and phc1 to specify HSPCs via modulation of Notch signalling.

Aminoacyl-tRNA synthetases (ARSs) have evolved to acquire various additional domains. These domains allow ARSs to communicate with other cellular proteins in order to promote non-translational functions. Vertebrate cytoplasmic isoleucyl-tRNA synthetases (IARS1s) have an uncharacterized unique domain, UNE-I. Here, we present the crystal structure of the chicken IARS1 UNE-I complexed with glutamyl-tRNA synthetase 1 (EARS1). UNE-I consists of tandem ubiquitin regulatory X (UBX) domains that interact with a distinct hairpin loop on EARS1 and protect its neighboring proteins in the multi-synthetase complex from degradation. Phosphomimetic mutation of the two serine residues in the hairpin loop releases IARS1 from the complex. IARS1 interacts with BRCA1 in the nucleus, regulates its stability by inhibiting ubiquitylation via the UBX domains, and controls DNA repair function.