Nuclear Functions of Nucleolin through Global Proteomics and Interactomic Approaches

Salvetti, Anna; Couté, Yohann; Epstein, Alberto L.; Arata, Loredana; Kraut, Alexandra; Navratil, Vincent; Bouvet, Philippe; Greco, Anna

doi:10.1021/acs.jproteome.6b00126

Cited by 53 publications

(56 citation statements)

References 49 publications

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“…To gain more insight into the potential implication of Ncl in spliceosome assembly/function, in this work, we studied the subcellular co-localization and interaction of Ac-Ncl with a component of catalytically activated/active spliceosome, P-SF3b155 (phosphorylated SF3B1). Together with the re-analysis of the splicing components found in our previous proteomic data [23], our data suggest that Ncl interact mostly with the pre-catalytic B complex and could probably participate in the assembly of the pre-catalytic spliceosomes.…”

Section: Introductionsupporting

confidence: 79%

“…Gene ontology of the purified proteins identify also 45 proteins implicated in RNA metabolism (ribosomal RNA excluded) including RNA stability, RNA nuclear export, pre-mRNA splicing. Interestingly, most of these proteins were still associated with Ncl after RNAse treatment, suggesting a direct protein-protein association [23].…”

Section: Introductionmentioning

confidence: 99%

“…A nucleolin interactome study using a Tap-tag purification of nuclear Ncl complexes allowed the identification of about 140 potential Ncl nuclear interacting partners [23]. About 50% of these proteins are involved in ribosome biogenesis which reflect the major nucleolar role of Ncl.…”

Section: Introductionmentioning

confidence: 99%

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Nucleolin Interacts and Co-Localizes with Components of Pre-Catalytic Spliceosome Complexes

Ugrinova¹,

Chalabi-Dchar²,

Monier³

et al. 2019

Sci

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Nucleolin is an RNA binding protein that is involved in many post-transcriptional regulation steps of messenger RNAs in addition to its nucleolar role in ribosomal RNA transcription and assembly in pre-ribosomes. Acetylated nucleolin was found to be associated with nuclear speckles and to co-localize with the splicing factor SC35. Previous nuclear pull down of nucleolin identified several splicing components and factors involved in RNA polymerase II transcription associated with nucleolin. In this report, we show that these splicing components are specifics of the pre-catalytic A and B spliceosomes, while proteins recruited in the Bact, C and P complexes are absent from the nucleolin interacting proteins. Furthermore, we show that acetylated nucleolin co-localized with P-SF3B1, a marker of co-transcriptional active spliceosomes. P-SF3B1 complexes can be pulled down with nucleolin specific antibodies. Interestingly, the alternative splicing of Fibronectin at the IIICS and EDB sites was affected by nucleolin depletion. These data are consistent with a model where nucleolin could be a factor bridging RNA polymerase II transcription and assembly of pre-catalytic spliceosome similarly to its function in the co-transcriptional maturation of pre-rRNA.

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Section: Introductionsupporting

confidence: 79%

Section: Introductionmentioning

confidence: 99%

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Nucleolin Interacts and Co-Localizes with Components of Pre-Catalytic Spliceosome Complexes

Ugrinova¹,

Chalabi-Dchar²,

Monier³

et al. 2019

Sci

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“…Nucleolin (NCL) is a highly conserved, multifunctional RNA binding protein (RBP) implicated in several cellular processes such as chromatin remodeling [1], rRNA synthesis [2], mRNA processing [3], ribosome assembly, and nucleo-cytoplasmic transport [4]. Elevated levels of NCL are present in highly proliferative cells including a variety of tumors [5][6][7][8][9].…”

Section: Introductionmentioning

confidence: 99%

Nucleolin phosphorylation regulates PARN deadenylase activity during cellular stress response

et al. 2017

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Nucleolin (NCL) is an abundant stress-responsive, RNA-binding phosphoprotein that controls gene expression by regulating either mRNA stability and/or translation. NCL binds to the AU-rich element (ARE) in the 3'UTR of target mRNAs, mediates miRNA functions in the nearby target sequences, and regulates mRNA deadenylation. However, the mechanism by which NCL phosphorylation affects these functions and the identity of the deadenylase involved, remain largely unexplored. Earlier we demonstrated that NCL phosphorylation is vital for cell cycle progression and proliferation, whereas phosphorylation-deficient NCL at six consensus CK2 sites confers dominant-negative effect on proliferation by increasing p53 expression, possibly mimicking cellular DNA damage conditions. In this study, we show that NCL phosphorylation at those CK2 consensus sites in the N-terminus is necessary to induce deadenylation upon oncogenic stimuli and UV stress. NCL-WT, but not hypophosphorylated NCL-6/S*A, activates poly (A)-specific ribonuclease (PARN) deadenylase activity. We further demonstrate that NCL interacts directly with PARN, and under non-stress conditions also forms (a) complex (es) with factors that regulate deadenylation, such as p53 and the ARE-binding protein HuR. Upon UV stress, the interaction of hypophosphorylated NCL-6/S*A with these proteins is favored. As an RNA-binding protein, NCL interacts with PARN deadenylase substrates such as TP53 and BCL2 mRNAs, playing a role in their downregulation under non-stress conditions. For the first time, we show that NCL phosphorylation offers specificity to its protein-protein, protein-RNA interactions, resulting in the PARN deadenylase regulation, and hence gene expression, during cellular stress responses.

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“…Proteins were stacked in a single band in the top of a SDS-PAGE gel (4-12% NuPAGE, Life Technologies) and stained with Coomassie blue R-250 before in-gel digestion using modied trypsin (Promega, sequencing grade) as previously described [37].…”

Section: Sample Preparation and Nanolc-ms/ms Analysesmentioning

confidence: 99%

Beyond target-decoy competition: stable validation of peptide and protein identifications in mass spectrometry-based discovery proteomics

Couté

Bruley

Bürger

2019

Preprint

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Target-decoy competition (TDC) is the most popular method for false discovery rate (FDR) control in bottom-up discovery proteomics. Despite unquestionable statistical foundations, we unveil a so far unknown weakness of TDC: its intrinsic lack of stability vis-à-vis practical conditions of application. Although some consequences of this instability have already been empirically described, they may have been misinterpreted. This work pinpoints evidence that TDC will become less reliable along with improved accuracies of modern mass spectrometers. We therefore propose to replace TDC by a totally different method to control the FDR at spectrum, peptide and protein levels, while benefiting from the theoretical guarantees of Benjamini-Hochberg framework. This method being simpler to use, faster to compute and more stable than TDC, we argue that it is more adapted to the standardization and throughput constraints of nowadays proteomic platforms.

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Nuclear Functions of Nucleolin through Global Proteomics and Interactomic Approaches

Cited by 53 publications

References 49 publications

Nucleolin Interacts and Co-Localizes with Components of Pre-Catalytic Spliceosome Complexes

Nucleolin Interacts and Co-Localizes with Components of Pre-Catalytic Spliceosome Complexes

Nucleolin phosphorylation regulates PARN deadenylase activity during cellular stress response

Beyond target-decoy competition: stable validation of peptide and protein identifications in mass spectrometry-based discovery proteomics

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