Project description:Motor unit remodelling involving repeated denervation and re-innervation occurs throughout life. The efficiency of this process declines with age contributing to neuromuscular deficits. We investigated differentially expressed genes (DEG) in muscle following peroneal nerve crush to model of motor unit remodelling in C57Bl6 mice. Muscle RNA was isolated at 3 days post-crush, RNA libraries were generated using poly-A selection, sequenced.
Project description:Temperature, as a universal enviromental factor, has prolonged effect on physiological and pathological functions of different species. In order to expolore the temperol effect of temperature on C.elegans longevity, we used microarray to check the whole-genome expression profiling of L4 larvae and Day3-old adults of C.elegans maintaining at different temperature Adult worms are collected after maintained at 25 degree and 15 degree for 3days from later L4 stage, while Larva worms are collected after growing at 25 degree and 15 degree from embryo to late L4 stege. Then total RNA are extracted with TRI reagent(Life Technologies) from~120 adults or ~300 larvae. The concentrations and quality of total RNA are checked with Thermo Nanodrop 2000c(Thermo Scientific,WilminGton,DE) and Agilent 2100 BioAnalyzer (Agilent Technologies, Palo Alto, CA). Total RNA samples were hybrydized with Affymetrix C. elegans Gene 1.1 ST Array Strip. Preparation of cDNA, hybridization, quality controls and scanning of arrays were performed according to the manufacturer's protocol (Affymetrix, Santa Clara, CA) at the microarray core facility of University of Michigan .
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:Regulation of gene expression is linked to the organization of the genome. With age, chromatin alterations occur on all levels of genome organization, accompanied by changes in the gene expression profile. However, little is known about the changes on the level of transcriptional regulation. Here, we used a multi-omics approach and integrated ATAC-, RNA- and NET-seq to identify age-related changes in the chromatin landscape of murine liver and to investigate how these are linked to transcriptional regulation. We provide the first systematic inventory of the connection between aging, chromatin accessibility and transcriptional regulation in a whole tissue. Aging in murine liver is characterized by an increase in chromatin accessibility at promoter regions, but not in an increase of transcriptional output. Instead, aging is accompanied by a decrease of promoter-proximal pausing of RNA polymerase II (Pol II). We propose that these changes in transcriptional regulation are due to a reduced stability of the pausing complex and may represent a mechanism to compensate for the age-related increase in chromatin accessibility in order to prevent aberrant transcription.
Project description:Biliary tract carcinoma (BTC) has a poor prognosis due to limited treatment options. There is therefore urgent need to identify new targets and to design innovative therapeutic approaches. Among potential candidate molecules, we evaluated the non-receptor tyrosine kinase Src, observing promising antitumor effects of its small molecule inhibitor Saracatinib in BTC preclinical models. The presence of an active Src protein was investigated by immunohistochemistry in 19 surgical samples from BTC patients. Upon Saracatinib treatment, the phosphorylation of Src and of its downstream transducers was evaluated in the BTC cell lines TFK-1, EGI-1, HuH28 and TGBC1-TKB. The effect of Saracatinib on proliferation and migration was analyzed in these same cell lines, and its antitumor activity was essayed in EGI-1 mouse xenografts. Saracatinib-modulated transcriptome was profiled in EGI-1 cells and in tumor samples of the xenograft model. Src was activated in about 80% of the human BTC samples. In cultured BTC cell lines, low-dose Saracatinib counteracted the activation of Src and of its downstream effectors, increased the fraction of cells in G0/G1 phase, and inhibited cell migration. At high concentrations (median dose from 2.26 to 6.99 µM), Saracatinib was also capable of inhibiting BTC cell proliferation. In vivo, Saracatinib treatment resulted in delayed tumor growth, associated with an impaired vascular network. We here provide a demonstration that the targeted inhibition of Src kinase by Saracatinib is of therapeutic benefit in preclinical models of BTC. We propose our results as a basis for the design of Saracatinib-based clinical applications. EGI-1 cell line treated with Saracatinib at the dose of 10 µM vs EGI-1 cell line untreated; EGI-1 xenograft treated with Saracatinib at the dose of 25 mg/Kg/die vs EGI-1 xenograft untreated Transcriptional alteration mediated by Saracatinib in vitro and in vivo
Project description:Here, we present the crystal structure of the catalytic domain of M5 bound to two magnesium ions, which reveals the role of each catalytic site acidic residue in metal binding. We further use hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) to investigate the possibility of structural rearrangements that would allow cleavage of the 5’-strand, and use small-angle X-ray scattering (SAXS) to study the M5 binding to the pre-5S rRNA substrate. This study provides new details on the M5 mechanism used for cleavage of the dsRNA substrate, which proceeds via two metal ions and structural rearrangements of both M5 and the pre-5S rRNA substrate.
Project description:As a newly identified mRNA modification, the regulation of ac4C remains largely unexplored. RNA-binding proteins (RBPs) that specifically binds to ac4C modification and mediate downstream cellular activities (readers) have not been reported yet. We synthesized acetylated and non-acetylated RNA probes by in vitro transcription. The sequences of the probes were segments of FUS and 18s rRNA, which contain ac4C sites as reported. A biotin-RNA pulldown assay and mass spectrometry were performed with HEK 293T cell lysates.
Project description:Transcriptional response of rat dental pulp cells (DPCs) cultured with SAHA at early and late mineralisation time points Transcript profiling of DPC identified several novel genes expression induced and supressed by HDACi at 24 hrs and 14 days under mineralising conditions. SAHA induces several members of the MMP family of endopepsidases (TIMP-1, MMP-9, MMP-13) and other members of the endochondral ossification pathway at 24 h. 8 experiemental parameters were analysed, each carried out in quadruplicate
Project description:Extremophilic archaea employ diverse chemical RNA modifications, providing a rich source of new enzymes for biotechnologically valuable RNA manipulations. Our understanding of the modified nucleoside profiles in Archaea, as well as the functions and dynamic regulation of specific RNA modifications is far from complete. Here, we established an extensive profile of nucleoside modifications in thermophilic and mesophilic Archaea through highly sensitive LC-MS/MS analysis and rigorous non-coding RNA depletion, identifying - with high confidence - at least four previously unannotated modifications in archaeal mRNAs. Nucleoside quantification analysis conducted on total, large, small, and mRNA-enriched subfractions of the model hyperthermophilic archaeon Thermococcus kodakarensis revealed a series of modifications whose abundance is dynamically responsive to growth temperatures, implying that specific RNA modifications are fitness relevant under specific growth conditions. To predict the RNA-modifying enzymes most likely to generate the new and dynamic RNA modifications, we leveraged a bioinformatics analysis of open-access databases to annotate likely functional domains of archaeal proteins. Putative enzyme activities were confirmed in vitro and in vivo by assessing the presence of the target RNA modification in genetic deletion strains of T. kodakarensis. Our approach led to the discovery of a methyltransferase-encoded gene responsible for m7G modification in the P-loop of 23S rRNA peptidyl transferase center. This novel finding validates an effective platform for discovering RNA-modifying enzymes through LC-MS/MS analysis that will accelerate efforts of the community towards uncovering the complex and dynamic roles of RNA modifications.
Project description:Pseudomonas plecoglossicida is a facultative pathogen that is associated with diseases of multiple fish, mainly at 15-20 °C. Although fish disease caused by P. plecoglossicida has led to significant economic losses, the mechanisms of the temperature-dependent virulence are unclear. Here, we try to identify potential pathogenicity mechanisms and demonstrate the direct regulation of virulence factors by temperature with iTRAQ.