Dataset Information

Dynamic regulation of the COP9 signalosome: Insights into the spatial localization and subunit versatility of the complex

ABSTRACT: HeLa cells were fracitonated into nuclear soluble, chromatin bound and cytosolic. Extracts were immunoprecipitated with a CSN3 antibody, then eluted with glycine. Proteins were digested, then enriched for phosphopeptides. Samples were analyzed on an LTQ-Orbitrap. Data was analyzed using the TRansOMics software for time alignment and feature detection. MS/MS spectra searched using Mascot.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Hek-293t Cell

SUBMITTER: Yishai Levin

LAB HEAD: Michal Sharon

PROVIDER: PXD000277 | Pride | 2019-11-25

REPOSITORIES: Pride

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Dynamic regulation of the COP9 signalosome in response to DNA damage.

Füzesi-Levi Maria G MG Ben-Nissan Gili G Bianchi Elisabetta E Zhou Houjiang H Deery Michael J MJ Lilley Kathryn S KS Levin Yishai Y Sharon Michal M

Molecular and cellular biology 20140113 6

The COP9 signalosome (CSN) is an evolutionarily conserved protein complex that participates in the regulation of the ubiquitin/26S proteasome pathway by controlling the function of cullin-RING-ubiquitin ligases. Impressive progress has been made in deciphering its critical role in diverse cellular and developmental processes. However, little is known about the underlying regulatory principles that coordinate its function. Through biochemical and fluorescence microscopy analyses, we determined th ...[more]

PMID: 24421388

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Project description:Tino is an A+U-Rich Element (ARE) binding protein first identified through its ability to bind to bcl-2 mRNA and to contribute to its degradation. It has recently been recognized as a shorter form of the human Mex-3D protein (hMex-3D), one of the four members of the family of Mex-3 RNA-binding phosphoproteins. In C. elegans, ceMex-3 is a translational regulator that plays a key role in early embryonic development and in the maintenance of worm germ line totipotency. To examine the potential functional conservation between ceMex-3 and hMex3, we have used complementary microarray-based approaches to identify mRNAs directly bound to Tino/hMex-3D. Computational analysis of these target mRNAs resulted in the identification of an U-rich, 34- to 39-nucleotide long, consensus, forming loops of variable sizes. Remarkably, more than half of Tino/hMex-3D targets also contain the consensus for Quaking, which is the human ortholog of GLD-1, a regulator of nematode gametogenesis. All together, our results suggest that Tino/hMex-3D belongs to a regulatory circuit of mRNA trans-acting factors involved in cell fate and differentiation. Keywords: RIP-chip and (recombinant)RIP-Chip analysis of Tino/hMEX-3D mRNPs To identify the transcripts directly bound in vivo by Tino/hMex-3D, we have used two complementary strategies. First, using Tino-his transfected HEK293 cells, we have analysed the RNAs immunoprecipitated by Tino/hMex-3D (RNA-IP complexes). Second, we have developed an in vitro nitrocellulose RNA-protein binding assay using a truncated form of Tino, TinoΔRING-his. First for the in vivo approach, we have transiently transfected HEK293 cells with a His-tagged Tino expressing construct (see Materials) and isolated Tino target mRNAs by immunoprecipitation (IP) assays carried out under conditions preserving mRNA-protein complex integrity. Second, for the in vitro approach, cRNAs were prepared from a human placenta cDNA library and incubated with TinoΔRING-his. This deleted form of Tino-his protein conserves its mRNA binding activity. mRNAs directly bound to TinoΔRING-his protein were subsequently isolated using a nitrocellulose RNA-protein binding assay. A supplementary file of ordered genes based on z-ratios, derived from experimental and negative control samples for each Affymetrix probe, is appended below.

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