Long noncoding RNA NORAD promotes epithelial mesenchymal transition progression in lung adenocarcinoma
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE159348 | GEO | 2023/10/10
REPOSITORIES: GEO
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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: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: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:Long noncoding RNAs (lncRNAs) can regulate the activity of interacting RNA binding proteins (RBPs), but how lncRNAs efficiently compete against other transcripts to bind and regulate RBP activity is poorly understood. This problem is exemplified by NORAD, a lncRNA that maintains genomic stability by inhibiting PUMILIO (PUM) proteins. Here we show that NORAD is able to out-compete other PUM-binding transcripts by nucleating the formation of phase-separated PUM condensates, termed NP bodies. Dual mechanisms of PUM recruitment, involving multivalent PUM-NORAD and PUM-PUM interactions, enable NORAD to sequester a super-stoichiometric amount of PUM in these condensates. Accordingly, synthetic RNAs that induce PUM phase separation rescue genome instability in NORAD-deficient cells. These results illuminate mechanisms of RBP regulation by RNA-driven phase separation and uncover an essential role for this process in genome maintenance.
2021-03-25 | GSE154812 | GEO
Project description:Purpose: PUMILIO proteins are known to repress target genes by specifically binding to PUMILIO response elements (PREs) in target mRNAs. NORAD is a noncoding RNA that negatively regulates PUMILIO activity. The goal of this study was to determine the gene expression changes that result from knockout of NORAD or overexpression of PUMILIO and to test whether NORAD knockout causes PUMILIO hyperactivity. Methods: RNA-seq libraries were prepared using the TruSeq Stranded Total RNA with Ribo-Zero Human/Mouse/Rat Sample Preparation kit (Illumina) and sequenced using the 100 bp paired-end protocol on an Illumina HiSeq 2000. For comparing NORAD+/+ and NORAD-/- HCT116 cells, 3 biological replicates per genotype were sequenced. For PUM overexpression experiments, 3 replicates of GFP-expressing HCT116 cells (negative control) and 2 independent PUM1- or PUM2-overexpressing clones (2 replicates each) were sequenced. Results: Gene expression profiles show that PUMILIO target genes are downregulated in both NORAD knockout cells and PUMILIO overexpressing cells. Conclusions: These data indicate that NORAD sequesters PUMILIO, preventing excessive repression of PUMILIO target genes that are important for maintaining genomic stability.
2015-12-03 | GSE75440 | 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:MiR-33a is involved in the maintenance of Glioma Initiating Cells (GIC) and tumor progression. MicroRNA-33a could promote GIC growth and self-renewal by regulating two pathways including cAMP/PKA pathway and Notch pathway. We used microarrays to identify the direct target genes of miR-33a in a glioblastoma cell line D456MG. We used microarrays to detail the global change of gene expression after miR-33a over-expression and identified target genes during this process. Cells were infected with lenti-virus expressing a control vector (pWPXLD) or human primary miR-33a. Then RNA was extracted and gene expression was profiled by Affymetrix microarray.
2014-07-17 | E-GEOD-59484 | biostudies-arrayexpress
Project description:MiR-33a is involved in the maintenance of Glioma Initiating Cells (GIC) and tumor progression. MicroRNA-33a could promote GIC growth and self-renewal by regulating two pathways including cAMP/PKA pathway and Notch pathway. We used microarrays to identify the direct target genes of miR-33a in a glioblastoma cell line D456MG. We used microarrays to detail the global change of gene expression after miR-33a over-expression and identified target genes during this process.
2014-07-17 | GSE59484 | GEO
Project description:In this study, we aimed to investigate the functional roles of miR-33a in PCa.We investigated the relative miR-33a level in normal and tumor prostate samples as well as PCa cell lines. Then, we performed a detailed functional analysis of mir-33a in LNCaP and VCaP PCa cells and evaluated proliferative, invasive and anchorage independent growth potential of cells upon overexpression and knockdown of miR-33a. We next explored the potential direct targets of miR-33a in PCa cells via utilizing gene expression microarray analysis, bioinformatics search, further qRT-PCR, western blot, and luciferase assay confirmation. Our results demonstrated that miR-33a is significantly downregulated in PCa tumor samples and PCa cell lines, pointing its tumor suppressor potential in PCa. Overexpression and knockdown of miR-33a significantly altered the proliferative, invasive and anchorage independent growth potentials of cells through altering the expression of its direct target PIM1. Ectopic induction of MiR-33a expression reversed the impacts of PIM1 overexpression on cellular phenotypes associated with PCa progression. Our results suggest that mir-33a exerts its tumor suppressor potential through targeting its direct target PIM1 and carries crucial roles in PCa tumorigenesis.
2017-10-31 | GSE89034 | GEO