Gene expression changes in human pluripotent and fibroblast cells and mouse embryonic fibroblasts upon NORAD lncRNA knockdown
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1007599 | ENA |
REPOSITORIES: ENA
Similar Datasets
Project description:RNA sequencing data from knockdowns of the long non-coding RNA NORAD in human foreskin fibroblasts (HFFs), human embryonic stem cells (hESCs), and mouse embryonic fibroblasts (MEFs) for varying durations (8–72 hours, depending on the cell type). Total RNA was purified using TRI-reagent, and libraries were prepared using the CEL-seq2 protocol. RNA-seq data were processed with cutadapt, aligned to the reference genome using RNA-STAR, and gene expression counts were generated with HTSEQ count.
2024-12-22 | GSE241264 | GEO
Project description:Thousands of long non-coding RNAs (lncRNAs) have been identified in the human genome, but specific biological functions and biochemical mechanisms have been discovered for only about a dozen lncRNAs. One specific lncRNA, Non-coding RNA Activated by DNA Damage (NORAD), has recently been shown by genetic deletion to be required for maintaining genomic stability, but its molecular mechanism is unknown. Here, we combine RNA antisense purification (RAP) and quantitative mass spectrometry to identify proteins that directly interact with NORAD in living cells. We show that NORAD interacts with proteins involved in DNA replication and repair in steady-state cells and localizes to the nucleus upon stimulation with replication stress or DNA damage. In particular, NORAD interacts with RBMX (an emerging component of the DNA-damage response) and encodes the strongest RBMX-binding site in the transcriptome. We demonstrate that NORAD controls the ability of RBMX to assemble a ribonucleoprotein complex, which we term NORAD-Activated Ribonucleoprotein Complex 1 (NARC1), containing known suppressors of genomic instability: topoisomerase I (TOP1), ALYREF and the PRPF19/CDC5L complex. Cells depleted of NORAD or RBMX display an increased frequency of chromosome segregation errors, reduced replication-fork velocity and altered cell cycle progression phenotypes that are mechanistically linked to TOP1 and PRPF19/CDC5L function. Expression of NORAD in trans can rescue defects caused by NORAD depletion, but rescue is significantly impaired when the RBMX-binding site in NORAD is deleted. Our results demonstrate that the interaction between NORAD and RBMX is important for NORAD function and that NORAD is required for the assembly of a previously unknown topoisomerase complex (NARC1) that contributes to maintaining genomic stability. Moreover, we uncover a novel function for lncRNAs in modulating the ability of an RNA-binding protein to assemble a higher-order ribonucleoprotein complex.
2018-08-27 | GSE114946 | GEO
Project description:Thousands of long non-coding RNAs (lncRNAs) have been identified in the human genome, but specific biological functions and biochemical mechanisms have been discovered for only about a dozen lncRNAs. One specific lncRNA, Non-coding RNA Activated by DNA Damage (NORAD), has recently been shown by genetic deletion to be required for maintaining genomic stability, but its molecular mechanism is unknown. Here, we combine RNA antisense purification (RAP) and quantitative mass spectrometry to identify proteins that directly interact with NORAD in living cells. We show that NORAD interacts with proteins involved in DNA replication and repair in steady-state cells and localizes to the nucleus upon stimulation with replication stress or DNA damage. In particular, NORAD interacts with RBMX (an emerging component of the DNA-damage response) and encodes the strongest RBMX-binding site in the transcriptome. We demonstrate that NORAD controls the ability of RBMX to assemble a ribonucleoprotein complex, which we term NORAD-Activated Ribonucleoprotein Complex 1 (NARC1), containing known suppressors of genomic instability: topoisomerase I (TOP1), ALYREF and the PRPF19/CDC5L complex. Cells depleted of NORAD or RBMX display an increased frequency of chromosome segregation errors, reduced replication-fork velocity and altered cell cycle progression phenotypes that are mechanistically linked to TOP1 and PRPF19/CDC5L function. Expression of NORAD in trans can rescue defects caused by NORAD depletion, but rescue is significantly impaired when the RBMX-binding site in NORAD is deleted. Our results demonstrate that the interaction between NORAD and RBMX is important for NORAD function and that NORAD is required for the assembly of a previously unknown topoisomerase complex (NARC1) that contributes to maintaining genomic stability. Moreover, we uncover a novel function for lncRNAs in modulating the ability of an RNA-binding protein to assemble a higher-order ribonucleoprotein complex.
2018-08-27 | GSE115429 | GEO
Project description:The number of known long noncoding RNA (lncRNA) functions is rapidly growing, but how those functions are encoded in their sequence and structure remains poorly understood. NORAD is a recently characterized, abundant, and highly conserved cytoplasmic lncRNA that is required for proper mitotic divisions in human cells. NORAD antagonizes repressors from the Pumilio family that bind at least 17 sites spread through 12 repetitive units in NORAD sequence. Here we study conserved sequences in NORAD repeats, identify additional interacting partners, and characterize the interaction between NORAD and the RNA binding protein SAM68 (KHDRBS1), which is required for NORAD function in antagonizing Pumilio. The interactions between NORAD, Pumilio and SAM68 provide a paradigm for how specific repeated and structured elements with a lncRNA can facilitate its function.
2019-01-01 | GSE104856 | GEO
Project description:Although numerous long noncoding RNAs (lncRNAs) have been identified, our understanding of their roles in mammalian physiology remains limited. Here we investigated the physiologic function of the conserved lncRNA Norad in vivo. Deletion of Norad in mice results in genomic instability and mitochondrial dysfunction, leading to a dramatic multi-system degenerative phenotype resembling premature aging. Loss of tissue homeostasis in Norad-deficient animals is attributable to augmented activity of PUMILIO proteins, which act as post-transcriptional repressors of target mRNAs to which they bind. Norad is the preferred RNA target of PUMILIO2 (PUM2) in mouse tissues and, upon loss of Norad, PUM2 hyperactively represses key genes required for mitosis and mitochondrial function. Accordingly, enforced Pum2 expression fully phenocopies Norad deletion, resulting in rapid-onset aging-associated phenotypes. These findings provide new insights and open new lines of investigation into the roles of noncoding RNAs and RNA binding proteins in normal physiology and aging.
2019-02-05 | GSE121688 | GEO
Project description:Although numerous long noncoding RNAs (lncRNAs) have been identified, our understanding of their roles in mammalian physiology remains limited. Here we investigated the physiologic function of the conserved lncRNA Norad in vivo. Deletion of Norad in mice results in genomic instability and mitochondrial dysfunction, leading to a dramatic multi-system degenerative phenotype resembling premature aging. Loss of tissue homeostasis in Norad-deficient animals is attributable to augmented activity of PUMILIO proteins, which act as post-transcriptional repressors of target mRNAs to which they bind. Norad is the preferred RNA target of PUMILIO2 (PUM2) in mouse tissues and, upon loss of Norad, PUM2 hyperactively represses key genes required for mitosis and mitochondrial function. Accordingly, enforced Pum2 expression fully phenocopies Norad deletion, resulting in rapid-onset aging-associated phenotypes. These findings provide new insights and open new lines of investigation into the roles of noncoding RNAs and RNA binding proteins in normal physiology and aging.
2019-02-05 | GSE121684 | GEO
Project description:We used Targeted RNase H-mediated Extraction of crosslinked RBPs (TREX)to assess the endogenous binding partners of the ND4 segment of NORAD long noncoding RNA (lncRNA) in human HCT116 cells. Extracted proteins from RNase H digested and control cells (4 replicate per region per condition) were compared, using label-free (LFQ) Quantitative proteomics.
2023-12-27 | PXD045385 | Pride
Project description:Long non-coding RNAs (lncRNAs) play important roles in regulating diverse biological processes including DNA damage and repair. We have recently reported that the DNA damage inducible lncRNA DNA damage-sensitive RNA1 (DDSR1) regulates DNA repair by homologous recombination (HR). Since lncRNAs also modulate gene expression, we identified gene expression changes upon DDSR1 knockdown in human fibroblast cells. Gene expression analysis after RNAi treatment targeted against DDSR1 revealed 119 genes that show differential expression. Here we provide a detailed description of the microarray data (NCBI GEO accession number GSE67048) and the data analysis procedure associated with the publication by Sharma et al., 2015 in EMBO Reports [1].
| S-EPMC4664778 | biostudies-literature
Project description:We describe here the proteome of the human breast cancer cell line MDA-MB-231 expressing normal levels of the lncRNA NORAD, or after knock-down NORAD with siRNA and LNA gapmers.
2023-08-28 | PXD039920 | Pride
Project description:Mass spectrometry data were processed using MSFragger via FragPipe with default parameters. For downstream analysis, Perseus was used. The MaxLFQ intensity values were log2-transformed and filtered to include only proteins detected in at least two replicates. Missing values were imputed from a normal distribution. Statistical analysis was performed using a two-sample t-test, with a permutation-based FDR applied (S0 parameter = 0.1)
2024-12-23 | PXD056160 | Pride