Project description:Comparison between WT Stm1, deltaN terminal and control AP-MS. The pulldown was performed with 3 biological indipendent replicates.
Project description:A long-standing question in developmental and reproductive biology is when the mammalian embryo becomes sufficiently distinct from its oocyte precursor. Myriads of studies examined the messenger RNAs that change during the oocyte-to-embryo transition, whereas proteins have been much less studied, in spite of their greater vicinity to phenotype. In the present study we modified the widely used embryo culture medium KSOM (PMID 12470333, PMID 10859270) to make it apt for our application. We replaced the serum albumin with polyvinylpyrrolidone and also replaced the natural Arginine and Lysine with their “heavy” isotopic variants Arginine 13C 15N and Lysine 13C 15N. Fertilized oocytes were retrieved from oviducts of gonadotropin-primed B6C3F1 females mated to CD1 males, and cultured at 37 degrees Celsius under 5% CO2 in KSOM containing 0.3 mM Arginine 13C 15N and 0.2 mM Lysine 13C 15N, which are the regular concentrations of these two amino acids in KSOM medium (PMID 12470333; PMID 10859270). After 4 days of culture, the embryos of the isotopic group had undergone blastocyst formation just like the control embryos cultured in normal medium. Samples of approx. 500 “heavy”-labeled blastocysts were collected zona-free and subjected to mass spectrometric analysis. The median labeling rate was 83%, ranging from 0% in proteins that did not incorporate any Arginine 13C 15N and Lysine 13C 15N, to 100% in proteins that were completely labeled. Our study demonstrates that a commonly used, chemically defined medium can be adapted for Stable Isotope Labeling by/with Amino acids in Cell culture (SILAC) and combined with high-resolution mass spectrometry, in a preimplantation embryo setting. This allows to tackle long-standing questions in developmental and reproductive biology, such as the identification of putative maternal (0% labeled), putative embryonic (100% labeled) or shared proteins in live mammalian embryos.
Project description:Commercial human heart whole tissue lysates were analyzed with LC-MS/MS with an inclusion list of alternative sequences (Lau et al. bioRxiv 2019).
Project description:We measured global RNA levels using RNA-seq in cas3 E. coli cells with intact CRISPR arrays or cells with the CRISPR-I array deleted to determine if off-target events driven by endogenous spacers affect local gene expression.
Project description:We measured global RNA levels using RNA-seq in cas3+ E. coli cells with intact CRISPR arrays or cells with the CRISPR-I array deleted to determine if off-target events driven by endogenous spacers affect RNA levels.
Project description:The aim of the experiment was to determine differences in RNA levels between wild-type E. coli and E. coli with an hns deletion. We used RNA-seq, but to control for variation in sample preparation and sequencing, we combined each E. coli RNA sample with a spike-in Salmonella RNA sample of known concentration. Libraries constructed in duplicate from strains grown with and without Salmonella spike-in to assess absolute genome-wide changes in transcription.This allowed us to normalise RNA levels between samples during data analysis.
Project description:Protein ubiquitination is an essential process that rapidly regulates protein synthesis, function, and fate in dynamic environments. Among its non-proteolytic functions, K63 ubiquitin accumulates in yeast cells exposed to oxidative stress, stalling ribosomes at elongation. K63 ubiquitin conjugates accumulate because of redox inhibition of the deubiquitinating enzyme Ubp2, however, the role and regulation of ubiquitin conjugating enzymes in this pathway remained unclear. Here we found that the E2 Rad6 binds and modifies elongating ribosomes during oxidative stress. We elucidated a mechanism by which Rad6 and its human homolog UBE2A are redox-regulated by forming reversible disulfides with the E1 activating enzyme, Uba1. We further showed that Rad6 activity is necessary to regulate translation, antioxidant defense, and adaptation to stress. Finally, we showed that Rad6 is required to induce phosphorylation of the translation initiation factor eIF2α, providing a novel link for K63 ubiquitin, elongation stalling, and the integrated stress response.