Abstract
AU-rich elements (AREs) in the 3'-UTR of unstable transcripts play a vital role in the regulation of many inflammatory mediators. To identify novel ARE-dependent gene regulators, we screened a human leukocyte cDNA library for candidates that enhanced the activity of a luciferase reporter bearing the ARE sequence from TNF (ARE(TNF)). Among 171 hits, we focused on Zfand5 (zinc finger, AN1-type domain 5), a 23-kDa protein containing two zinc finger domains. Zfand5 expression was induced in macrophages in response to IFNγ and Toll-like receptor ligands. Knockdown of Zfand5 in macrophages decreased expression of ARE class II transcripts TNF and COX2, whereas overexpression stabilized TNF mRNA by suppressing deadenylation. Zfand5 specifically bound to ARE(TNF) mRNA and competed with tristetraprolin, a protein known to bind and destabilize class II ARE-containing RNAs. Truncation studies indicated that both zinc fingers of Zfand5 contributed to its mRNA-stabilizing function. These findings add Zfand5 to the growing list of RNA-binding proteins and suggest that Zfand5 can enhance ARE-containing mRNA stability by competing with tristetraprolin for mRNA binding.
Highlights
AU-rich elements (AREs) in the 3Ј-UTR of mRNA confer instability; the mechanisms are incompletely understood
Our study reveals that Zfand5 stabilizes class II ARE-RNAs by binding directly to the ARE-RNA and competing with tristetraprolin (TTP), a zinc finger-containing protein that destabilizes mRNAs with Class II AREs
We used a similar strategy with a chimeric luciferase reporter construct containing the ARE sequence of TNF (ARETNF) and observed that luciferase activity in RAW cells was greatly reduced as compared with cells carrying the same reporter with 3Ј-UTR from actin [26]
Summary
AU-rich elements (AREs) in the 3Ј-UTR of mRNA confer instability; the mechanisms are incompletely understood. Zfand bound to ARETNF mRNA and competed with tristetraprolin, a protein known to bind and destabilize class II ARE-containing RNAs. Truncation studies indicated that both zinc fingers of Zfand contributed to its mRNA-stabilizing function. We undertook an unbiased screen, using a leukocyte cDNA library, to identify genes whose expression led to enhanced activity of a luciferase reporter construct bearing an ARETNF within its 3Ј-UTR. Our study reveals that Zfand stabilizes class II ARE-RNAs by binding directly to the ARE-RNA and competing with tristetraprolin (TTP), a zinc finger-containing protein that destabilizes mRNAs with Class II AREs
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