Project description:The ground state of pluripotency is defined as a minimal unrestricted state as present in the Inner Cell Mass (ICM). Mouse embryonic stem cells (ESCs) grown in a defined serum-free medium with two kinase inhibitors (‘2i’) reflect this state, whereas ESCs grown in the presence of serum (‘serum’) share more similarities with post implantation epiblast cells. Pluripotency results from an intricate interplay between cytoplasmic, nuclear and chromatin-associated proteins. Therefore, quantitative information on the (sub)cellular proteome is essential to gain insight in the molecular mechanisms driving different pluripotent states. Here, we describe a full SILAC workflow and quality controls for proteomic comparison of 2i and serum ESCs. We demonstrate that this workflow is applicable for subcellular proteomics of the cytoplasm, nuclear and chromatin. The obtained quantitative information revealed increased levels of naïve pluripotency factors on the chromatin of 2i ESCs. Further, we demonstrate that these pluripotent states are supported by distinct metabolic programs, which include upregulation of free radical buffering by the glutathione pathway in 2i ESCs. Through induction of intracellular radicals, we show that the altered metabolic environment renders 2i ESCs less sensitive to oxidative stress. Altogether, this work provides novel insights into the proteome landscape underlying ground state pluripotency.
Project description:microRNAs (miRNAs) are a large class of small non-coding RNAs which post-transcriptionally regulate the expression of a large fraction of all animal genes and are important in a wide range of biological processes. Recent advances in high-throughput sequencing allow miRNA detection at unprecedented sensitivity, but the computational task of accurately identifying the miRNAs in the background of sequenced RNAs remains challenging. For this purpose we have designed miRDeep2, a substantially improved algorithm which identifies canonical and non-canonical miRNAs such as those derived from transposable elements and informs on high-confidence candidates that are detected in multiple independent samples. Analyzing data from seven animal species representing the major animal clades, miRDeep2 identified miRNAs with an accuracy of 98.6-99.9% and reported hundreds of novel miRNAs. To test the accuracy of miRDeep2, we knocked down the miRNA biogenesis pathway in a human cell line and sequenced small RNAs before and after. The vast majority of the >100 novel miRNAs expressed in this cell line were indeed specifically down-regulated, validating most miRDeep2 predictions. Last, a new miRNA expression profiling routine, low time and memory usage and user-friendly interactive graphic output can make miRDeep2 useful to a wide range of researchers." high-throughput sequencing was used to profile small RNA expression in a human MCF-7 cell line before and after Dicer knock-down
Project description:CDK9 is a critical kinase required for the productive transcription of protein-coding genes by RNA polymerase II (pol II) in higher eukaryotes. Phosphorylation of targets including the elongation factor SPT5 and the carboxyl-terminal domain (CTD) of RNA pol II allow the polymerase to pass an early elongation checkpoint (EEC), which is encountered soon after initiation. In addition to halting RNA polymerase II at the EEC, CDK9 inhibition also causes premature termination of transcription across the last exon, loss of polyadenylation factors from chromatin, and loss of polyadenylation of nascent transcripts. Inhibition of the phosphatase PP2A abrogates the premature termination and loss of polyadenylation caused by CDK9 inhibition, suggesting that CDK9 and PP2A, working together, regulate the coupling of elongation and transcription termination to RNA maturation. Our phosphoproteomic analyses, using either DRB or an analog-sensitive CDK9 cell line confirm the splicing factor SF3B1 as an additional key target of this kinase. CDK9 inhibition causes loss of interaction of splicing and export factors with SF3B1, suggesting that CDK9 also helps to co-ordinates coupling of splicing and export to transcription.
Project description:This study sought to determine the dynamic changes of miRNA expression during mouse granulopoiesis. We not only performed analyses of miRNA expression levels in whole cells but also analyzed purified nuclear and cytoplasmic cell fractions to profile miRNA subcellular localization. qRT-PCR analysis of miRNAs was performed on whole cell, nuclear and cytoplasmic RNAs extracted from mouse hemopoietic stem cells (LSKs), promyelocytes, myelocytes and granulocytes. 100 ng of RNA was reversed transcribed using the Taqman miRNA Reverse Transcription Kit and Megaplex RT Primers rodent pool A and B (Life Technologies). Complementary DNA (cDNA) was amplified using a TaqMan rodent microRNA A and B Array v2.0 (Life Technologies) with TaqMan Universal PCR Master Mix on an ABI 7900HT Sequence Detection System.
Project description:To combat virus infections, which are major human killers, a deeper understanding of how viruses reprogram their hosts to create optimal production of progeny is needed. Most knowledge on the regulation of cellular gene expression during adenovirus infection is derived from studies of mRNA expression. Here, we investigated the changes in cellular protein expression during the early phase of adenovirus type 2 (Ad2) infection of primary human cells by stable isotope labeling in cell culture (SILAC) with subsequent liquid chromatography-high resolution tandem mass spectrometric (LC-MS/MS) analysis using a Q-Exactive Orbitrap instrument. Cells were in-depth evaluated 6 and 12 hours post infection (hpi) and two biological replicates were investigated using swapped labeling. In total, 2027 and 2150 proteins were quantified at 6 and 12 hpi, respectively. Among them, 431 and 544 were deregulated more than 1.5-fold at the two time points. For the deregulated proteins the change in protein expression was compared with that of late phase of infection (see PXD004095). Pathway analysis showed that De novo purine and pyrimidine biosynthesis, Glycolysis and Cytoskeletal regulation by Rho GTPase pathways are activated early during the infection, while the inactivation of the Integrin signalling pathway starts between 6 and 12 hpi. The transcription factor MYC was predicted to be activated with time, and the phosphopeptide analysis revealed the up-regulation of phosphosites related with glycolysis or cytoskeletal reorganization. These results complement the previous knowledge obtained from transcriptomic data, and show novel and specific aspects of how adenovirus influence host cell gene expression at the protein level.The results contribute to our understanding of host cell gene regulation during infection at a deeper level.
Project description:Differentially expressed genes were determined by single-end sequencing (stranded protocol) following Trim24 and/or p53 knock down in the mouse ES cells grown in 2i media. Triplicates were generated for treatment and control samples but for p53 knock down (duplicate).
Project description:Analysis of HeLa cells at 24 hours after transfection with wild type miR-1, miR-124, miR-181 versus control transfected HeLa cells. Results were compared to protein down-regulation at 48 hours measured by SILAC-MS. Analysis of HeLa cells at 24 hours after transfection with wild type miR-1, miR-124, miR-181 versus control transfected HeLa cells. Results were compared to protein down-regulation at 48 hours measured by SILAC-MS.
Project description:The Sertoli cells (Sc) of 5 days (infant) and 12 days (pubertal) old rat were isolated and cultured in triplicates. Nuclear and cytoplasmic fractionation was done for both the cases. All the four protein fractions (nuclear and cytoplasm of infant and pubertal Sc) were anaysed using Lc-MS/MS. SWATH analysis was done for all the four samples in biological and technical replicates. The objective was to quantity the proteins of all the samples with respect to each other at whole proteome level.
Project description:The Sertoli cells (Sc) of 5 days (infant) and 12 days (pubertal) old rat were isolated and cultured in triplicates. Nuclear and cytoplasmic fractionation was done for both the cases. All the four protein fractions (nuclear and cytoplasm of infant and pubertal Sc) were anaysed using Lc-MS/MS.