Ribosome profiling on polysomes extracted via sucrose gradient and IP
ABSTRACT: Technical feasibility study to determine data quality and reproducibilty. Ribosome profiling was performed by either sucrose gradient (SG) or immunoprecipitation (IP). 1 sucrose gradient sample, 2 immunoprecipitation samples in two concentrations of beads
Project description:SUM159PT cells were grown either in vitro (in culture) or in vivo (mouse), after which RPL10a tagged with GFP was used to perform extraction by immoprecipitation and subsequent ribosome profiling Two batches of in vitro grown cells, each having 2 replicates. Two batches of in vivo grown cells from tumors grown in two individual mice, each having tumors on left (first batch) and right side (second batch). Extraction and ribosome profiling was done independently for the two batches of samples.
Project description:Ribosome profiling on sections taken from a kidney tumor For two tumors: 2 sections of normal tissue and 4 samples of tumor tissue, for another two tumors 1 section of normal tissue and 1 section of tumor tissue
Project description:Four different breast cancer cell lines were grown either under unrestricted growth conditions or in medium lacking glutamine, and ribosome profliling was performed on material extracted from these samples 1 replicate per condition
Project description:MCF10A were either untreated, treated with torin or by starvation, to induce autophagy. RNA sequencing and ribosome profiling was performed. 1 biological replicate for each condition
Project description:These are the results of the iCLIP experiment for p62/SQSTM1 in Human Huh-7 cells treated with DMSO. We used iCLIP method to identify the RNA targets of p62 and nucleotide positions of the p62 interaction on RNA. We used 2 replicates and 2 different antibodies against endogenous p62 to enrich protein/RNA complexes. cDNAs were tagged with iCLIP composite barcodes (e.g. NNNTTGTNN) which contain 4 sample-encoding bases (e.g. TTGT) and and 5 random bases (noted with N in NNNTTGTNN example) which serve as unique molecular identifiers to post-filter PCR duplicates. These composite barcodes are found in the read headers (after last colon ':' character) of submitted fastq files.
Project description:The innate immune response is the first line of defense against pathogens, and factors that control this cellular response represent targets for treating both infectious and inflammatory diseases. Here, we reveal a novel role for the human helicase SETX, also previously implicated in amyotrophic lateral sclerosis (ALS4) and ataxia with oculomotor apraxia (AOA2), in controlling the magnitude of the antiviral response. Cells depleted for SETX and AOA2 patient-derived SETX-null cells show increased expression of antiviral mediators in response to infection. Mechanistically, this effect is achieved through SETX-mediated inhibition of RNAPII transcription of antiviral genes, and depends on SETX helicase activity. Our results suggest that SETX helps maintain the delicate balance between controlling viral infection and avoiding the potentially detrimental effects of an excessive antiviral response. More broadly, the observation that SETX can regulate the transcriptional activity of specific genes may have important implications for disorders where SETX function is compromised. A549 cells that were transfected with either control non-targeting or SETX-specific siRNAs were infected with the Influenza A virus (A/PR/8/34(ΔNS1) strain) at a multiplicity of infection (MOI) of 3 for 4 hours. Nuclei were then extracted and used to prepare Global Run-On Sequencing (GRO-seq) libraries.
Project description:To understand the impact of alternative translation initiation on a proteome, we performed the first study on protein turnover using positional proteomics and ribosome profiling to distinguish between N-terminal proteoforms of individual genes. Overall, we monitored the stability of 1,941 human N-terminal proteoforms, including 147 N-terminal proteoform pairs that originate from alternative translation initiation, alternative splicing or incomplete processing of the initiator methionine. Ribosome profiling of lactimidomycin and cycloheximide treated human Jurkat T-lymphocytes
Project description:Native RNA immunoprecipitation of Cnot7-bound transcripts. Abstract: Accumulating evidence supports the role of an aberrant transcriptome as a driver of tumor cell metastatic potential. Deadenylation is a general regulatory node for post-transcriptional control by microRNAs and other determinants of RNA stability. We show here that CCR4-NOT subunit CNOT7 is a deadenylase-dependent driver of tumor cell autonomous metastatic potential. Metastasis promotion by CNOT7 is dependent on contact with CNOT1 and TOB1. We further show TOB1 independently drives metastasis. RNA-immunoprecipitation and integrated transcriptome wide analyses reveal that CNOT7-regulated transcripts are enriched for a tripartite 3’UTR motif bound by RNA-binding proteins known to complex with CNOT7, TOB1, and CNOT1. Collectively, our data support a model of CNOT7, TOB1, CNOT1, and RNA-binding proteins collectively exerting post-transcriptional control on a metastasis suppressive transcriptional program to drive tumor cell metastasis. 48 total samples, 4-5 biological replicates, two forms of control: input samples and vector controls