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A Novel Role for Nucleolin in Splice Site Selection

ABSTRACT: In search for nuclear partners of initiator-tRNA, previously shown to be involved in a quality control of splice site selection, we identified nucleolin (NCL) as specifically and directly bound to it in the nucleus and not in the cytoplasm (using affinity purification of UV crosslinked nuclear initiator-tRNA followed by mass spectrometry). To further explore the role of NCL in splice site selection, we knocked down NCL. We report the activation of 399 latent splice sites upon NCL knockdown, as revealed by RNA deep sequencing of siRNA treated NCL compared to control siRNA. Our study identifies NCL as the first protein component of this quality control mechanism of splice site selection. Our study thus establishes an experimental strategy and computational pipeline that could be used to identify other components of this quality control mechanism.

ORGANISM(S): Homo sapiens

PROVIDER: GSE142836 | GEO | 2022/03/08

REPOSITORIES: GEO

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A Novel Role for Nucleolin in Splice Site Selection

Project description:A Novel Role for Nucleolin in Splice Site Selection

| PRJNA598591 | ENA

Analysis of Nucleolin depletion impact on the KRAS-induced proteomic changes in mouse Pancreatic ductal adenocarcinoma cells.

Project description:To reveal the role of Nuclelin (Ncl) downstream of mutant KRAS in shaping the proteome of Pancreatic Ductal Adenocarcinoma (PDAC) cells, we carried out a quantitative proteomics analysis of tumour cells isolated from an inducible mouse model of PDAC (iKras PDAC) (Ying et al., 2012). In this model, oncogenic Kras (G12D) expression can be controlled by administration of doxycycline (Dox). Tandem Mass Tagging (TMT) was employed for quantitative analysis of Dox induced (24hrs) vs. non-induced iKras-PDAC cells, from control or Nucleolin depleted settings. Two independent siRNA oligos were used against Ncl. All samples barcoded and pooled together using TMT 6plex labelling kit (Thermo).

2023-02-12 | PXD038338 | Pride

Binding of Nucleolin to HBV cccDNA

Project description:Covalently closed circular DNA (cccDNA) forms the basis for replication and persistence of hepatitis B virus (HBV) in the chronically infected liver. In this study we sought to identify host factors interacting with the HBV genome. Nucleolin (Ncl) was identified as a potential interactor of HBV cccDNA. This interaction was veriefied using an established ChIPseq protocol. The data show that Ncl binds cccDNA albeit at lower levels than HBcAg. Ncl deposition occurs across the genome without a clear localization and a variable pattern of deposition between experiments. This verifies the interaction of Ncl with HBV-cccDNA.

2021-12-31 | GSE123715 | GEO

Nuclear nucleolin interactome analysis

Project description:Nucleolin (NCL) is a major component of the cell nucleolus, which has the ability to rapidly shuttle to several other cell’s compartments. NCL has been shown to play important roles in a variety of essential functions among which ribosome biogenesis, gene expression and cell growth. However, the precise mechanisms underlying NCL functions are still unclear. Our study aimed to provide new information on NCL functions via the identification of its nuclear interacting partners. Using an interactomics approach, we identified 140 binding partners of NCL, among which, a 100 of them, were specifically found associated to NCL independently of the presence of RNA.

2016-04-11 | PXD003554 | Pride

Profiling of microRNAs associated with Nucleolin (NCL) in human pulmonary artery smooth muscle cells

Project description:Analysis of miRNAs associated with NCL via RNA immunoprecipitation in pulmonary artery smooth muscle cells using NCL antibodies to identify a distinct set of miRNAs regulated by NCL. Results provide insight into the regulatory mechanism of RNA binding proteins in vascular smooth muscle cells.

2023-03-20 | GSE184972 | GEO

Nucleolin-aptamer therapy in retinoblastoma: molecular changes and mass spectrometry–based imaging

Project description:Retinoblastoma (RB) is an intraocular childhood tumor which, if left untreated, leads to blindness and mortality. Nucleolin (NCL) protein which is differentially expressed on the tumor cell surface, binds ligands and regulates carcinogenesis and angiogenesis. We found that NCL is over expressed in RB tumor tissues and cell lines compared to normal retina. We studied the effect of nucleolin-aptamer (NCL-APT) to reduce proliferation in RB tumor cells. Aptamer treatment on the RB cell lines (Y79 and WERI-Rb1) led to significant inhibition of cell proliferation. Locked nucleic acid (LNA) modified NCL-APT administered subcutaneously (s.c.) near tumor or intraperitoneally (i.p.) in Y79 xenografted nude mice resulted in 26 and 65% of tumor growth inhibition, respectively. Downregulation of inhibitor of apoptosis proteins, tumor miRNA-18a, altered serum cytokines, and serum miRNA-18a levels were observed upon NCL-APT treatment. Desorption electrospray ionization mass spectrometry (DESI MS)-based imaging of cell lines and tumor tissues revealed changes in phosphatidylcholines levels upon treatment. Thus, our study provides proof of concept illustrating NCL-APT-based targeted therapeutic strategy and use of DESI MS-based lipid imaging in monitoring therapeutic responses in RB.

2016-11-15 | GSE89818 | GEO

Nucleolin HITS-CLIP, RNA-Seq and Ribo-Seq upon inhibition of 5'-tRFCys

Project description:Expression of 5'-tRFCys is significantly upregulated during breast cancer metastasis and suppression of 5'-tRFCys significantly reduced metastatic lung colonization of breast cancer. We identified Nucleolin (Ncl) as the direct binding partner of 5'-tRFCys. To identify the molecular mechanism underlying 5'-tRFCys promotion of breast cancer, we performed Ncl HITS-CLIP, RNA-Seq, Ribosome profiling (Ribo-Seq) upon inhibition of 5'-tRFCys. These analyses reveal that 5'-tRFCys enhances Ncl binding to a subset of transcripts, resulting in increased transcript stability of those transcripts and expression.

2022-06-03 | GSE173373 | GEO

Nucleolin uses its RBD2 domain to recognize long-loop G-Quadruplexes from human minisatellite CEB25

Project description:We investigated herein the interaction between nucleolin (NCL) and a set of G4 sequences derived from the CEB25 human minisatellite which adopt a parallel topology while differing by the length of the central loop (from 9nt to1nt). It is revealed that NCL strongly binds to long-loop (9-5 nt) G4 whilst interacting weakly with the shorter variants (loop < 3nt). Photocrosslinking experiments using 5-bromouracil (BrdU) modified sequences further confirmed the loop-length dependency thereby indicating that the CEB25-WT (9nt) is the best G4 substrate. Quantitative proteomic analysis (LC-MS/MS) of the photocrosslinking product(s) obtained with NCL bound to this sequence enabled the identification of one contact site within the 9nt loop. The protein fragment identified is located in the helix of the RBD2 domain of NCL, shedding light on the role of this structural element in the G4-loop recognition. Then, the ability of a panel of benchmark G4 ligands to prevent the NCL/G4 interaction was explored. It was found that only the most potent ligand PhenDC3 is able to inhibit NCL binding, thereby suggesting that the terminal guanine quartet is also a strong determinant of G4 recognition, putatively through interaction with the RGG domain. This study puts forward the molecular mechanism by which NCL recognizes G4-containing long loops and leads to propose a model implying a concerted action of RBD2 and RGG domains to achieve specific G4 recognition via a dual loop-quartet interaction.

2020-03-19 | PXD016529 | Pride

iCLIP Analysis of WT, phospho-defective, and phospho-mimicking mutants of mouse Nucleolin in iKras PDAC cells

Project description:This experiment aimed to identify Nucleolin (Ncl) binding sites on a transcriptome-wide scale, by performing iCLIP in mouse iKras PDAC cells (Ying et al., 2012) transiently transfected with a mammalian expression construct encoding a wild-type (WT) myc-Ncl, a phospho-defective mutant in which S28, S34, S40, and S41 residues were mutated to A, or a phospho-mimicking mutant with these residues mutated to D. Cells transfected with an empty vector (myc-pRK5-DEST) were used as controls in the experiment.

2023-03-01 | E-MTAB-12481 | biostudies-arrayexpress

Convergence of Cockayne Syndrome Group A and B Proteins at rRNA Transcription through Nucleolin Regulation

Project description:Cockayne Syndrome (CS) is a rare neurodegenerative disease characterized by short stature, cachexia, sun-sensitivity, accelerated aging, and short lifespan. Mutations in two human genes, ERCC8/CSA and ERCC6/CSB, are causative for CS and the protein products of these genes, CSA and CSB, while structurally unrelated, play roles in DNA repair and other aspects of DNA metabolism in human cells. Many clinical and molecular features of CS remain poorly understood, and it has been suggested that CSA and CSB regulate transcription of rDNA genes and ribosome biogenesis. The goal of this study was to investigate the dysregulation of rRNA synthesis in CS. Here, we report that Nucleolin (Ncl), a nucleolar protein that regulates rRNA synthesis and ribosome biogenesis, interacts specifically with CSA and CSB. In addition, CSA induces ubiquitination of Ncl, enhances binding of CSB to Ncl, and CSA and CSB both stimulate binding of Ncl to rDNA and subsequent rRNA synthesis. These findings suggest that CSA and CSB are positive regulators of rRNA synthesis via Ncl regulation. A majority of CS patients carry mutations in CSA and CSB and present with similar clinical features, thus our findings may provide novel insights into disease mechanism and the neuropathological features of CS.

2020-03-16 | GSE122736 | GEO

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