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Molecular and Cellular Biology, November 2005, p. 9520-9531, Vol. 25, No. 21
0270-7306/05/$08.00+0     doi:10.1128/MCB.25.21.9520-9531.2005

Identification and Functional Outcome of mRNAs Associated with RNA-Binding Protein TIA-1

Isabel López de Silanes,1 Stefanie Galbán,1 Jennifer L. Martindale,1 Xiaoling Yang,1 Krystyna Mazan-Mamczarz,1 Fred E. Indig,2 Geppino Falco,3 Ming Zhan,2* and Myriam Gorospe1*

Laboratory of Cellular and Molecular Biology,1 Research Resources Branch,2 Laboratory of Genetics, National Institute on Aging-Intramural Research Program, National Institutes of Health, Baltimore, Maryland 212243

Received 15 May 2005/ Returned for modification 8 June 2005/ Accepted 8 August 2005

The RNA-binding protein TIA-1 (T-cell intracellular antigen 1) functions as a posttranscriptional regulator of gene expression and aggregates to form stress granules following cellular damage. TIA-1 was previously shown to bind mRNAs encoding tumor necrosis factor alpha (TNF-{alpha}) and cyclooxygenase 2 (COX-2), but TIA-1 target mRNAs have not been systematically identified. Here, immunoprecipitation (IP) of TIA-1-RNA complexes, followed by microarray-based identification and computational analysis of bound transcripts, was used to elucidate a common motif present among TIA-1 target mRNAs. The predicted TIA-1 motif was a U-rich, 30- to 37-nucleotide (nt)-long bipartite element forming loops of variable size and a bent stem. The TIA-1 motif was found in the TNF-{alpha} and COX-2 mRNAs and in 3,019 additional UniGene transcripts (~3% of the UniGene database), localizing preferentially to the 3' untranslated region. The interactions between TIA-1 and target transcripts were validated by IP of endogenous mRNAs, followed by reverse transcription and PCR-mediated detection, and by pulldown of biotinylated RNAs, followed by Western blotting. Further studies using RNA interference revealed that TIA-1 repressed the translation of bound mRNAs. In summary, we report a signature motif present in mRNAs that associate with TIA-1 and provide support to the notion that TIA-1 represses the translation of target transcripts.

* Corresponding author. Mailing address for Myriam Gorospe: Box 12, LCMB, NIA-IRP, NIH, 5600 Nathan Shock Dr., Baltimore, MD 21224. Phone: (410) 558-8443. Fax: (410) 558-8386. E-mail: myriam-gorospe{at}nih.gov. Mailing address for Ming Zhan (for bioinformatic inquiries): Bioinformatics Unit, RRB, NIA, NIH, 5600 Nathan Shock Dr., Baltimore, MD 21224. Phone: (410) 558-8373. Fax: (410) 558-8674. E-mail: zhanmi{at}mail.nih.gov.

Molecular and Cellular Biology, November 2005, p. 9520-9531, Vol. 25, No. 21
0022-538X/05/$08.00+0     doi:10.1128/MCB.25.21.9520-9531.2005




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