RESUMEN
Branched ubiquitin (Ub) chains constitute a sizable fraction of Ub polymers in human cells. Despite their abundance, our understanding of branched Ub function in cell signaling has been stunted by the absence of accessible methods and tools. Here we identify cellular branched-chain-specific binding proteins and devise approaches to probe K48-K63-branched Ub function. We establish a method to monitor cleavage of linkages within complex Ub chains and unveil ATXN3 and MINDY as debranching enzymes. We engineer a K48-K63 branch-specific nanobody and reveal the molecular basis of its specificity in crystal structures of nanobody-branched Ub chain complexes. Using this nanobody, we detect increased K48-K63-Ub branching following valosin-containing protein (VCP)/p97 inhibition and after DNA damage. Together with our discovery that multiple VCP/p97-associated proteins bind to or debranch K48-K63-linked Ub, these results suggest a function for K48-K63-branched chains in VCP/p97-related processes.
RESUMEN
During protein synthesis, charged tRNAs deliver amino acids to translating ribosomes, and are then re-charged by tRNA synthetases (aaRS). In humans, mutant aaRS cause a diversity of neurological disorders, but their molecular aetiologies are incompletely characterised. To understand system responses to aaRS depletion, the yeast glutamine aaRS gene (GLN4) was transcriptionally regulated using doxycycline by tet-off control. Depletion of Gln4p inhibited growth, and induced a GCN4 amino acid starvation response, indicative of uncharged tRNA accumulation and Gcn2 kinase activation. Using a global model of translation that included aaRS recharging, Gln4p depletion was simulated, confirming slowed translation. Modelling also revealed that Gln4p depletion causes negative feedback that matches translational demand for Gln-tRNAGln to aaRS recharging capacity. This maintains normal charged tRNAGln levels despite Gln4p depletion, confirmed experimentally using tRNA Northern blotting. Model analysis resolves the paradox that Gln4p depletion triggers a GCN4 response, despite maintenance of tRNAGln charging levels, revealing that normally, the aaRS population can sequester free, uncharged tRNAs during aminoacylation. Gln4p depletion reduces this sequestration capacity, allowing uncharged tRNAGln to interact with Gcn2 kinase. The study sheds new light on mutant aaRS disease aetiologies, and explains how aaRS sequestration of uncharged tRNAs can prevent GCN4 activation under non-starvation conditions.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Proteínas Serina-Treonina Quinasas/genética , ARN de Transferencia de Glutamina/genética , ARN de Transferencia/genética , Proteínas de Saccharomyces cerevisiae/genética , Aminoácidos/genética , Aminoácidos/metabolismo , Aminoacil-ARNt Sintetasas/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Homeostasis , Fosforilación , ARN de Transferencia de Glutamina/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Inanición/genética , Inanición/metabolismoRESUMEN
tRNA gene copy number is a primary determinant of tRNA abundance and therefore the rate at which each tRNA delivers amino acids to the ribosome during translation. Low-abundance tRNAs decode rare codons slowly, but it is unclear which genes might be subject to tRNA-mediated regulation of expression. Here, those mRNA targets were identified via global simulation of translation. In-silico mRNA translation rates were compared for each mRNA in both wild-type and a [Formula: see text] sup70-65 mutant, which exhibits a pseudohyphal growth phenotype and a 75% slower CAG codon translation rate. Of 4900 CAG-containing mRNAs, 300 showed significantly reduced in silico translation rates in a simulated tRNA mutant. Quantitative immunoassay confirmed that the reduced translation rates of sensitive mRNAs were [Formula: see text] concentration-dependent. Translation simulations showed that reduced [Formula: see text] concentrations triggered ribosome queues, which dissipated at reduced translation initiation rates. To validate this prediction experimentally, constitutive gcn2 kinase mutants were used to reduce in vivo translation initiation rates. This repaired the relative translational rate defect of target mRNAs in the sup70-65 background, and ameliorated sup70-65 pseudohyphal growth phenotypes. We thus validate global simulation of translation as a new tool to identify mRNA targets of tRNA-specific gene regulation.
Asunto(s)
Regulación de la Expresión Génica , Estudio de Asociación del Genoma Completo , Modelos Biológicos , Biosíntesis de Proteínas , ARN Mensajero/genética , ARN de Transferencia/genética , Alelos , Anticodón , Emparejamiento Base , Codón , Simulación por Computador , Dosificación de Gen , Regulación Fúngica de la Expresión Génica , Mutación , ARN Mensajero/metabolismo , ARN de Transferencia/metabolismo , Levaduras/genética , Levaduras/metabolismoRESUMEN
Gene expression can be regulated by a wide variety of mechanisms. One example concerns the growing body of evidence that the protein-production rate can be regulated at the level of translation elongation by controlling ribosome flux across the mRNA. Variations in the abundance of tRNA molecules cause different rates of translation of their counterpart codons. This, in turn, produces a variable landscape of translational rate across each and every mRNA, with the dynamic formation and deformation of ribosomal queues being regulated by both tRNA availability and the rates of translation initiation and termination. In the present article, a range of examples of tRNA control of gene expression are reviewed, and the use of mathematical modelling to develop a predictive understanding of the consequences of that regulation is discussed and explained. These findings encourage a view that predicting the protein-synthesis rate of each mRNA requires a holistic understanding of how each stage of translation, including elongation, contributes to the overall protein-production rate.