RESUMEN
The Elongin complex was originally identified as an RNA polymerase II (RNAPII) elongation factor and subsequently as the substrate recognition component of a Cullin-RING E3 ubiquitin ligase. More recent evidence indicates that the Elongin ubiquitin ligase assembles with the Cockayne syndrome B helicase (CSB) in response to DNA damage and can target stalled polymerases for ubiquitylation and removal from the genome. In this report, we present evidence that the CSB-Elongin ubiquitin ligase pathway has roles beyond the DNA damage response in the activation of RNAPII-mediated transcription. We observed that assembly of the CSB-Elongin ubiquitin ligase is induced not just by DNA damage, but also by a variety of signals that activate RNAPII-mediated transcription, including endoplasmic reticulum (ER) stress, amino acid starvation, retinoic acid, glucocorticoids, and doxycycline treatment of cells carrying several copies of a doxycycline-inducible reporter. Using glucocorticoid receptor (GR)-regulated genes as a model, we showed that glucocorticoid-induced transcription is accompanied by rapid recruitment of CSB and the Elongin ubiquitin ligase to target genes in a step that depends upon the presence of transcribing RNAPII on those genes. Consistent with the idea that the CSB-Elongin pathway plays a direct role in GR-regulated transcription, mouse cells lacking the Elongin subunit Elongin A exhibit delays in both RNAPII accumulation on and dismissal from target genes following glucocorticoid addition and withdrawal, respectively. Taken together, our findings bring to light a new role for the CSB-Elongin pathway in RNAPII-mediated transcription.
Asunto(s)
ADN Helicasas/genética , Enzimas Reparadoras del ADN/genética , Elonguina/genética , Proteínas de Unión a Poli-ADP-Ribosa/genética , ARN Polimerasa II/genética , Ubiquitina-Proteína Ligasas/genética , Animales , Síndrome de Cockayne/enzimología , Síndrome de Cockayne/genética , ADN Helicasas/química , ADN Helicasas/ultraestructura , Reparación del ADN/genética , Enzimas Reparadoras del ADN/química , Enzimas Reparadoras del ADN/ultraestructura , Elonguina/química , Elonguina/ultraestructura , Humanos , Ratones , Complejos Multiproteicos/química , Complejos Multiproteicos/genética , Complejos Multiproteicos/ultraestructura , Proteínas de Unión a Poli-ADP-Ribosa/química , Proteínas de Unión a Poli-ADP-Ribosa/ultraestructura , ARN Polimerasa II/química , Receptores de Glucocorticoides/química , Receptores de Glucocorticoides/genética , Ubiquitina/química , Ubiquitina/genética , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/ultraestructura , Ubiquitinación/genéticaRESUMEN
The budding yeast spindle pole body (SPB) is anchored in the nuclear envelope so that it can simultaneously nucleate both nuclear and cytoplasmic microtubules. During SPB duplication, the newly formed SPB is inserted into the nuclear membrane. The mechanism of SPB insertion is poorly understood but likely involves the action of integral membrane proteins to mediate changes in the nuclear envelope itself, such as fusion of the inner and outer nuclear membranes. Analysis of the functional domains of the budding yeast SUN protein and SPB component Mps3 revealed that most regions are not essential for growth or SPB duplication under wild-type conditions. However, a novel dominant allele in the P-loop region, MPS3-G186K, displays defects in multiple steps in SPB duplication, including SPB insertion, indicating a previously unknown role for Mps3 in this step of SPB assembly. Characterization of the MPS3-G186K mutant by electron microscopy revealed severe over-proliferation of the inner nuclear membrane, which could be rescued by altering the characteristics of the nuclear envelope using both chemical and genetic methods. Lipid profiling revealed that cells lacking MPS3 contain abnormal amounts of certain types of polar and neutral lipids, and deletion or mutation of MPS3 can suppress growth defects associated with inhibition of sterol biosynthesis, suggesting that Mps3 directly affects lipid homeostasis. Therefore, we propose that Mps3 facilitates insertion of SPBs in the nuclear membrane by modulating nuclear envelope composition.
Asunto(s)
Puntos de Control de la Fase M del Ciclo Celular/genética , Proteínas de la Membrana/genética , Microtúbulos/genética , Proteínas Nucleares/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Alelos , Proliferación Celular , Homeostasis , Metabolismo de los Lípidos , Proteínas de la Membrana/metabolismo , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Membrana Nuclear/genética , Membrana Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Estructura Terciaria de Proteína , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
The SARS-CoV-2 pandemic and vaccination campaign has illustrated the need for high throughput serological assays to quantitatively measure antibody levels. Here, we present a protocol for a high-throughput colorimetric ELISA assay to detect IgG antibodies against the SARS-CoV-2 spike protein. The assay robustly distinguishes positive from negative samples, while controlling for potential non-specific binding from serum samples. To further eliminate background contributions, we demonstrate a computational pipeline for fitting ELISA titration curves, that produces an extremely sensitive antibody signal metric for quantitative comparisons across samples and time.
RESUMEN
Irradiation of low enriched uranium-molybdenum fuel results in the production and agglomeration of fission gas bubbles that can potentially lead to fuel failure. Manual point volume fraction counting in accordance with ASTME562 standard has been historically used to conduct pore size distribution analysis. While effective, the manual methodology is not efficient and therefore not feasible for the characterization of several fuel plates in a timely manner. In this contribution, ImageJ and MATLAB software were investigated as suitable alternatives to manual counting. Validation and verification were performed to show that the results are reproducible. Image analysis revealed insignificant variation of fission gas pore morphology with fission density. In addition, the results from two different sample preparation techniques - vibratory polishing and focused ion beam milling were compared. Sample preparation has more than 1% influence on the results of pore size distribution analysis. Comprehensive comparison identified vibratory polishing as the preferred method for conducting fission gas pore size distribution analysis.