Genome-wide Analysis Identifies Interleukin-10 mRNA as Target of Tristetraprolin

Georg Stoecklin,Scott A Tenenbaum,Thomas Mayo,Sridar V Chittur,Ajish D George,Timothy E Baroni,Perry J Blackshear,Paul Anderson

doi:10.1074/jbc.m709657200

Abstract

Tristetraprolin (TTP) is an RNA-binding protein required for the rapid degradation of mRNAs containing AU-rich elements. Targets regulated by TTP include the mRNAs encoding tumor necrosis factor-alpha, granulocyte-macrophage colony-stimulating factor, interleukin-2 (IL-2), and immediate early response 3. To identify novel target mRNAs of TTP in macrophages, we used a genome-wide approach that combines RNA immunoprecipitation and microarray analysis. A list was compiled of 137 mRNAs that are associated with TTP with an estimated accuracy on the order of 90%. Sequence analysis revealed a highly significant enrichment of AU-rich element motifs, with AUUUA pentamers present in 96% and UUAUUUAUU nonamers present in 44% of TTP-associated mRNAs. We further show that IL-10 is a novel target regulated by TTP. IL-10 mRNA levels were found to be elevated because of a reduced decay rate in primary macrophages from TTP(-/-) mice. Our study demonstrates the importance of experimental approaches for identifying targets of RNA-binding proteins.

Highlights

Tristetraprolin (TTP)2 is an RNA-binding protein that recognizes AU-rich elements (AREs), which are characteristic for a group of short-lived mRNAs
Other possible targets of TTP were suggested by alternative approaches; knock down of TTP via RNA-interference showed that the rapid degradation of 1,4-galactosyltransferase 1, c-myc, cyclin D1, and vasculo-endothelial growth factor mRNA is dependent on TTP (8 –10)
Our results show that the AUUUA pentamer and the UUAUUUAUU nonamer are highly enriched in 3Ј-UTRs of TTP-associated mRNAs

Summary

EXPERIMENTAL PROCEDURES

RIP—RAW264.7 cells were lysed for 10 min on ice in RIP buffer containing 50 mM Tris, pH 8.0, 150 mM NaCl, 1 mM MgCl2, 1.0% Nonidet P-40, and 10% glycerol and freshly supplemented with 100 ␮M dithiothreitol, 1 mM sodium vanadate, 50 mM NaF, 20 nM okadaic acid, and EDTA-free complete protease inhibitors (Roche Applied Science). 50 ng of isolated RNA was converted to labeled fragmented cRNA using the Affymetrix small sample protocol (Version II). Digoxigenin-labeled RNA probes were generated by SP6 in vitro transcription using custom reagents (Roche Applied Science), and cDNA templates were generated with the following primer pairs: G314/G316 for TNF␣, G366/ G390 for ubiquitin specific peptidase 46 (Usp46), G370/G392 for Kelch-like protein 2 (Klhl2), G376/G395 for teratocarcinoma expressed protein (Tera), G380/G397 for AK166182, G382/G398 for TNF receptor-associated factor-2-binding protein (T2BP), G386/G400 for Gm561, and G388/G401 for eIF4Ebinding protein 1 (4EBP1). For pTet-7B-IL-10UTR, the murine IL-10 3Ј-UTR was amplified by reverse transcription-PCR using primers G192 and G193 and ligated as a BamHI - BglII fragment into the BglII site of pTet-7B. For pTet7B-RAN-UTR, the murine RAN 3Ј-UTR was amplified by reverse transcription-PCR using primers G228 and G229 and ligated as a NheI-BglII fragment into the NheI-BglII sites of pTet-7B-IL-10-UTR. MTT Assay—L929 cells were grown over night in 96-well plates at a density of 3.5 ϫ 104 cells per well, and medium containing recombinant mouse TNF␣ (554589, BD Pharmingen)

A TTP-associated mRNAs

RESULTS

DISCUSSION