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| Status |
Public on Feb 28, 2010 |
| Title |
RIP-chip analysis of Tino/hMEX-3D mRNPs |
| Organism |
Homo sapiens |
| Experiment type |
Other
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| Summary |
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
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| Overall design |
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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| Contributor(s) |
Minardi S, Venturini E, Lulli M, Donnini M, Lapucci A |
| Citation missing |
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| Submission date |
Jul 24, 2008 |
| Last update date |
Dec 06, 2018 |
| Contact name |
martino donnini |
| E-mail(s) |
martinod@unifi.it
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| Phone |
00390554598243
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| Fax |
00394598900
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| Organization name |
University of Florence
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| Department |
Pathology and Oncology
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| Lab |
Sergio Capaccioli
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| Street address |
viale GB Morgagni
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| City |
Firenze |
| State/province |
Firenze |
| ZIP/Postal code |
50134 |
| Country |
Italy |
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| Platforms (1) |
| GPL571 |
[HG-U133A_2] Affymetrix Human Genome U133A 2.0 Array |
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| Samples (4)
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| Relations |
| BioProject |
PRJNA113699 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE12239_RAW.tar |
9.2 Mb |
(http)(custom) |
TAR (of CEL) |
| GSE12239_z_ratio_data.txt |
1.2 Mb |
(ftp)(http) |
TXT |
| Processed data included within Sample table |
| Processed data are available on Series record |
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