Ribosome profiling of NEW1 knock-out of budding yeast Saccharomyces cerevisiae
ABSTRACT: New1 is not an essential gene but its deletion shows a cold-sensitive phenotype in yeast Saccharomyces cerevisiae. In this study, we compare the NEW1 knockout effect on translation using Ribo-Seq and RNA-Seq analyses.
Project description:Ribosome profiling (Ribo-Seq) and RNA-Seq analysis of eEF3 depletion in yeast (Saccharomyces cerevisiae). eEF3 depletion was induced by methionine in a modified strain where the native promoter was replaced by methionine repressible MET25 promoter. Conditional depletion enables us to study global effects of an essential gene.
Project description:Loss of New1 leads to a cold-sensitive phenotype of yeast Saccharomyces cerevisiae. In this study we investigated the effect of NEW1 knockout on translation using Ribo-Seq and RNA-Seq analyses.
Project description:To define what genes are predominantly or specifically expressed in either soma or germline in C. elegans adults, total RNA was extracted from germline-less glp-4 mutant animals or from dissected gonads, respectively. Total RNA sequencing was peformed in duplicates. Four samples in total.
Project description:Bone marrow (BM) cells were obtained by flushing the long bones of 8-week old C57BL/6 mice. BM cells were then were plated in macrophage SFM medium (Life Technologies) supplemented with penicillin-streptomycin and CSF-1 (Peprotech, 100 ng/ml) and cultured for one week to allow macrophage differentiation. BMDMs were polarized by adding IL-4 to the medium (40 ng/ml, Peprotech) for 72h or left untreated. 8 samples total, 2 groups, 4 biological replicates in each group
Project description:In this study, we make used of mRNA-seq and its ability to reliably quantify isoforms, integrating this data with ribosome profiling and LC-MS/MS, to assign ribosome footprints and peptides at the isoform level. We leverage the principle that most cell types, and even tissues, predominantly express a single principal isoform to set isoform-level mRNA-seq quantifications as priors to guide and improve allocation of footprints or peptides to isoforms. Through tightly integrated mRNAseq, ribosome footprinting and/or LC-MS/MS proteomics we demonstrate that a principal isoform can be identified in over 80% of gene products in homogenous HEK293 cell culture and over 70% of proteins detected in complex human brain tissue. Defining isoforms in experiments with matched RNA-seq and translatomic/proteomic data increases the functional relevance of such datasets and will further broaden our understanding of multi-level control of gene expression. In this PRIDE submission you will find the raw files for the HEK293 cell proteomics. Files for the human brain proteomics can be found at PXD005445. We have also uploaded a zip file that contains the input files for our HEK293 cell analysis, and the isoform level output files – there is a separate folder within the zip files for these. The data used to create the manuscript figures is in the Rdata file. Code for assigning peptides and footprints to isoforms can be found on Github here: https://github.com/rkitchen/EMpire
Project description:We carried out total RNA sequencing on RNA extracted from 5 brain subregions (cerebellum, cortex, hippocampus, hypothalamus and thalamus) and liver tissues of wild-type mice at 3 months of age. In total, two female and two male mice were used to obtain biological replicates for appropriate statistical analysis. Not only is this dataset used to perform expression analysis between brain subregions and between brain and liver, it is also used in-conjunction with 5hmC-seq methylation profiles that we performed to study the association between 5hmC and gene expression. Stranded RNA-seq library was prepared and sequenced on a HiSeq2500 by single end sequencing with 100 bp read length.
Project description:Current pharmacotherapies for symptomatic benign prostatic hyperplasia (BPH), an androgen receptor (AR) driven, inflammatory disorder affecting elderly men, include 5α-reductase (5AR) inhibitors (i.e. dutasteride and finasteride) to block the conversion of testosterone to the more potent AR ligand dihydrotestosterone (DHT). Since DHT is the precursor for estrogen receptor β (ERβ) ligands, 5AR inhibitors could potentially limit ERβ activation, which maintains prostate tissue homeostasis. We have uncovered signaling pathways in BPH-derived prostate epithelial cells (BPH-1) that are impacted by 5AR inhibition. The induction of apoptosis and repression of the cell-adhesion protein E-cadherin by the 5AR inhibitor, dutasteride, requires both ERβ and TGFβ. Dutasteride also induces cyclooxygenase type 2 (COX-2), which functions in a negative-feedback loop in TGFβ and ERβ signaling pathways as evidenced by the potentiation of apoptosis induced by dutasteride or finasteride upon pharmacological inhibition or shRNA-mediated ablation of COX-2. Concurrently, COX-2 positively impacts ERβ action through its effect on the expression of a number of steroidogenic enzymes in the ERβ-ligand metabolic pathway. Therefore, effective combination pharmacotherapies, which have included non-steroidal anti-inflammatory drugs, must take into account biochemical pathways affected by 5AR inhibition and opposing effects of COX-2 on the tissue protective action of ERβ. Next-generation sequencing (n=3) of shRNA mediated knockdown of COX-2 or scrambled control in BPH-1 prostate epithelial cell line
Project description:CHX is an inhibitor of translation elongation often used in ribosome profiling experiments. There is evidence that CHX treatment of cells may cause artefacts in the distribution of ribosomes on mRNAs. We investigate this possibility in S. pombe by performing ribosome profiling in the presence and absence of this drug.