The protein Zfand5 binds and stabilizes mRNAs with AU-rich elements in their 3'-untranslated regions

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.

FIGURE 1.

Identification and confirmation of Zfand5 as an ARE-RNA stabilizer. A, schematic display of the screening strategy (left). Right, representative results of primary, secondary, and final screens from one pool of cDNAs, expressed as -fold induction above the mean activity for the entire group. Green, E. coli culture; red, reporter activity-positive cDNA; yellow, reporter activity-negative cDNA. B, expression of Zfand5 resulted in enhanced reporter activities for luciferase reporter appended with ARE sequences. RAW cells were transfected with the indicated reporter constructs without (blank) or with an empty vector pCMV6 (mock) or pCMV6-Zfand5 (Zfand5). The results are calculated as the ratio of activities of firefly luciferase versus Renilla luciferase, expressed as -fold increase as compared with the blank and are mean ± S.E. (error bars) from three experiments. *, p < 0.01, Student's t test. C, expression of Zfand5 enhanced LPS-induced release of TNF and IL-6. RAW cells were transfected as in B without luciferase reporters for 24 h before the addition of 100 ng/ml LPS. TNF, IL-6, and IL-10 contents in the media were determined 24 h later by ELISA. *, p < 0.05, Student's t test. D, expression of Zfand5 retarded the decay of TNF mRNA. RAW cells were transfected as in B for 24 h, and then cells were treated with LPS (100 ng/ml) for 1 h before the addition of 10 μg/ml Act. D. RNA samples were prepared 0, 2, or 4 h thereafter. Transcripts for β-actin and TNF were determined by semiquantitative RT-PCR. Arrow, TNF amplicon; open arrowhead, β-actin amplicon. One of four similar experiments is shown.

FIGURE 2.

Induction of Zfand5 by inflammatory stimuli. A and B, Zfand5 induction by LPS. After exposure to LPS for 1 h (A) or to 100 ng/ml LPS for the indicated periods (B), cell-associated transcripts for GAPDH and Zfand5 were determined by semiquantitative RT-PCR. Integrated signals were normalized to that of GAPDH. The results are mean ± S.E. (error bars) from three experiments. *, p < 0.05 as compared with untreated samples, Student's t test. C, induction of Zfand5 in macrophages. RAW cells were exposed to LPS (100 ng/ml), poly(I:C) (1 μg/ml), CpG (2 μm), Pam3Cys (100 ng/ml), or IFNγ (100 units/ml) for 1 or 6 h. Transcripts for GAPDH and Zfand5 were determined and normalized as in A. The results are mean ± S.E. from four experiments.

FIGURE 3.

Knockdown of Zfand5 suppresses Class II ARE-containing transcripts. Knockdown of Zfand5 down-regulates TNF and COX2 mRNA. RAW cells were transfected with indicated shRNA for 24 h and then treated with 100 ng/ml Pam3Cys for 6 h. A, Zfand5 levels are shown with RT-PCR (mRNA) or Western blots (protein). B, the transcripts for TNF, COX2, NOS2, and p53 were assessed by semiquantitative RT-PCR. Right, quantification of the left panels. Integrated signals were normalized with respect to the β-actin (for mRNA) or α-tubulin (for protein) and expressed as the percentage of the average signal in CR1 and CR2. Results are mean ± S.E. (error bars) from three experiments.

FIGURE 4.

Zfand5 binds to ARE of TNF. A, sequences for the ARE^TNF and ARE^TNF mutant probes with mutated nucleotides in gray. B, Zfand5 binds specifically to ARE^TNF. Recombinant His-Zfand5 or BSA was incubated with 1 pmol of ARE^TNF or ARE^TNF mutant (ARE^TNF-mut) for 30 min at room temperature and fractionated on a native gel as described under “Experimental Procedures.” Supershift was demonstrated by incubating anti-His tag or control antibody with recombinant His-Zfand5 for 10 min before the addition of the probe. One of four similar experiments is shown.

FIGURE 5.

Zfand5 competes with TTP-ARE^TNF binding and reverses the destabilizing effects of TTP on TNF mRNA. A and B, Zfand5 competes with TTP-ARE^TNF binding. A (top), RNA EMSA. The numbers indicate the amounts of protein in μg. Open arrowheads, TTP-ARE complex; closed arrowheads, Zfand5-ARE complex. A (bottom), TTP or Zfand 5 in the EMSA were detected by Western blots. One of at least three similar experiments is shown. C, expression of Zfand5 reverses the destabilizing effects of TTP on TNF mRNA. RAW cells were transfected with TTP, Zfand5, or both for 24 h, and the stability of transcripts was determined as described in the legend to Fig. 1D 30 min after addition of Act. D. Results are mean ± S.E. from triplicates in one of two experiments.

FIGURE 6.

Zfand5 inhibits deadenylation of ARE-containing RNA and suppresses the deadenylation effects of TTP. A, RAW cells were transfected with vector (mock), Zfand5, or TTP for 24 h. Cytoplasmic extracts were incubated with A60 with (ARE) or without ARE (ΔARE) for different times before fractionation on 5% denaturing polyacrylamide gel. Deadenylation of the A60 was visualized using the Odyssey infrared imaging system (LI-COR Biotechnology) with a streptavidin-IRDye 800CW conjugate. Positions of A60 and A0 are indicated. B, quantification of results in A, expressed as percentage of A60 remaining at each time point. C, cytoplasmic extracts from cells transfected with vector (mock), Zfand5 (Zf5), TTP, or TTP with increasing amounts of Zfand5 were assayed for the deadenylation activity as in A. One of three similar experiments is shown.

FIGURE 7.

Both zinc fingers of Zfand5 contribute to its ARE-RNA-stabilizing function. A, schematic model of Zfand5 truncation mutants. B, Coomassie Blue stain of full-length Zfand5 and its truncation mutants with deletion of A20 (Z5ΔA20), AN1 (Z5ΔAN1), or both (Z5ΔA20ΔAN1) on SDS-PAGE. C, interaction of Zfand5 or its mutants with RNA probe ARE^TNF. RNA EMSA analysis of binding of Zfand5 and its mutants to ARE^TNF was performed as described in the legend to Fig. 4B. The open arrowhead indicates the Z5ΔAN1-ARE complex. D, deletion of zinc fingers affects the ability of Zfand5 to stabilize TNF mRNA. RAW cells expressing the indicated constructs were treated as in Fig. 1D. Transcripts were determined by semiquantitative RT-PCR. Arrow, TNF; open arrowhead, β-actin. One of three similar experiments is shown.