Eukaryotic arginylation is an essential post-translational modification that both modulates protein stability and regulates protein half-life through the N-degron pathway. Arginylation is catalyzed by a family of enzymes known as the arginyl-tRNA transferases (ATE1s), which are conserved across the eukaryotic domain. Despite its conservation and importance, little is known regarding the structure, mechanism, and regulation of ATE1s. In this work, we have discovered that ATE1s bind a previously unknown iron-sulfur cluster that is conserved across evolution. We have extensively characterized the nature of this iron-sulfur cluster, and we show that the presence of the iron-sulfur cluster is linked to alterations in arginylation efficacy. Finally, we demonstrate that the ATE1 iron-sulfur cluster is oxygen sensitive, which could be a molecular mechanism of the N-degron pathway to sense oxidative stress. Thus, our data provide the framework of a cluster-based paradigm of ATE1 regulatory control.
Mutations in the RNA helicase, DDX3X, are a leading cause of Intellectual Disability and present as DDX3X syndrome, a neurodevelopmental disorder associated with cortical malformations and autism. Yet the cellular and molecular mechanisms by which DDX3X controls cortical development are largely unknown. Here, using a mouse model of Ddx3x loss-of-function we demonstrate that DDX3X directs translational and cell cycle control of neural progenitors, which underlies precise corticogenesis. First, we show brain development is highly sensitive to Ddx3x dosage; Complete Ddx3x loss from neural progenitors causes microcephaly in females, whereas hemizygous males and heterozygous females show reduced neurogenesis without marked microcephaly. In addition, Ddx3x loss is sexually dimorphic, as its paralog, Ddx3y, compensates for Ddx3x in the developing male neocortex. Using live imaging of progenitors, we show that DDX3X promotes neuronal generation by regulating both cell cycle duration and neurogenic divisions. Finally, we use ribosome profiling in vivo to discover the repertoire of translated transcripts in neural progenitors, including those which are DDX3X-dependent and essential for neurogenesis. Our study reveals invaluable new insights into the etiology of DDX3X syndrome, implicating dysregulated progenitor cell cycle dynamics and translation as pathogenic mechanisms.
Eukaryotic arginylation is an essential post-translational modification that modulates protein stability and regulates protein half-life. Arginylation is catalyzed by a family of enzymes known as the arginyl-tRNA transferases (ATE1s), which are conserved across the eukaryotic domain. Despite their conservation and importance, little is known regarding the structure, mechanism, and regulation of ATE1s. In this work, we show that ATE1s bind a previously undiscovered [Fe-S] cluster that is conserved across evolution. We characterize the nature of this [Fe-S] cluster and find that the presence of the [Fe-S] cluster in ATE1 is linked to its arginylation activity, both in vitro and in vivo, and the initiation of the yeast stress response. Importantly, the ATE1 [Fe-S] cluster is oxygen-sensitive, which could be a molecular mechanism of the N-degron pathway to sense oxidative stress. Taken together, our data provide the framework of a cluster-based paradigm of ATE1 regulatory control.
Arginyltransferases (ATE1s) are a class of essential eukaryotic enzymes that catalyze arginylation, the posttranslational transfer of Arg from an aminoacylated tRNA to a range of protein targets. This modification typically occurs at N-terminal acidic residues, though ATE1 can also covalently attach Arg to mid-chain residues. Arginylation may have either degradative or non-degradative effects. For example, some arginylated proteins are processed through the Arg N-degron pathway, which marks these post-translationally modified proteins for degradation by the ubiquitin-proteasome system. In contrast, arginylation may also manifest non-degradative effects such as thermodynamic stability, subcellular relegalization, or functional changes. The diversity of ATE1's targets confers its role as a global regulator, influencing functions such as cardiovascular development, neurological processing, and even the stress response. However, a lack of ATE1 structural knowledge has limited the determination of its three-dimensional fold, how it is regulated, and how it recognizes its substrates. Using a combination of X-ray crystallography, cryo-EM, and size-exclusion chromatography-coupled small angle X-ray scattering (SEC-SAXS), our lab has successfully solved the structure of Saccharomyces cerevisiae ATE1 (ScATE1). The three-dimensional structure of ScATE1 reveals a bilobed protein containing a canonical GCN5-related Nacetyltransferase (GNAT) fold. Structural superpositions and electrostatic analyses indicate this domain as the location of catalytic activity and tRNA binding. Furthermore, the structure reveals the spatial connectivity of the Nterminal domain, which we previously showed binds an [Fe-S] cluster, to the enzymatic active site, hinting at the cluster's regulatory influence. As the first atomic-level structure of any ATE1, this achievement brings us closer to answering pressing questions regarding the molecular-level mechanism of eukaryotic post-translational arginylation.
Eukaryotic post-translational arginylation, mediated by the family of enzymes known as the arginyltransferases (ATE1s), is an important post-translational modification that can alter protein function and even dictate cellular protein half-life. Multiple major biological pathways are linked to the fidelity of this process, including neural and cardiovascular developments, cell division, and even the stress response. Despite this significance, the structural, mechanistic, and regulatory mechanisms that govern ATE1 function remain enigmatic. To that end, we have used X-ray crystallography to solve the first crystal structure of ATE1 from Saccharomyces cerevisiae ATE1 (ScATE1) to 2.85 Å resolution. The three-dimensional structure of ScATE1 reveals a bilobed protein containing a GCN5-related N-acetyltransferase (GNAT) fold, and this crystalline behavior is faithfully recapitulated in solution based on size-exclusion chromatography-coupled small angle X-ray scattering (SEC-SAXS) analyses and cryo-EM 2D class averaging. Structural superpositions and electrostatic analyses indicate this domain as the location of catalytic activity and tRNA binding, and these comparisons strongly suggest a mechanism for post-translational arginylation. Additionally, our structure reveals the spatial connectivity of the N-terminal domain, which we have previously shown to bind a regulatory [Fe-S] cluster, and the enzymatic active site, hinting at the atomic-level details of the cluster’s regulatory influence. When taken together, these insights into the first structure of ATE1 bring us closer to answering pressing questions regarding the molecular-level mechanism of eukaryotic post-translational arginylation.
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