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. 2012 Feb 17;287(8):5459-71.
doi: 10.1074/jbc.M111.312652. Epub 2011 Dec 27.

Direct binding of specific AUF1 isoforms to tandem zinc finger domains of tristetraprolin (TTP) family proteins

Vishram P Kedar  1 , Beth E Zucconi, Gerald M Wilson, Perry J Blackshear
Affiliations

Direct binding of specific AUF1 isoforms to tandem zinc finger domains of tristetraprolin (TTP) family proteins

Vishram P Kedar et al. J Biol Chem. .

Abstract

Tristetraprolin (TTP) is the prototype of a family of CCCH tandem zinc finger proteins that can bind to AU-rich elements in mRNAs and promote their decay. TTP binds to mRNA through its central tandem zinc finger domain; it then promotes mRNA deadenylation, considered to be the rate-limiting step in eukaryotic mRNA decay. We found that TTP and its related family members could bind to certain isoforms of another AU-rich element-binding protein, HNRNPD/AUF1, as well as a related protein, laAUF1. The interaction domain within AUF1p45 appeared to be a C-terminal "GY" region, and the interaction domain within TTP was the tandem zinc finger domain. Surprisingly, binding of AUF1p45 to TTP occurred even with TTP mutants that lacked RNA binding activity. In cell extracts, binding of AUF1p45 to TTP potentiated TTP binding to ARE-containing RNA probes, as determined by RNA gel shift assays; AUF1p45 did not bind to the RNA probes under these conditions. Using purified, recombinant proteins and a synthetic RNA target in FRET assays, we demonstrated that AUF1p45, but not AUF1p37, increased TTP binding affinity for RNA ∼5-fold. These data suggest that certain isoforms of AUF1 can serve as "co-activators" of TTP family protein binding to RNA. The results raise interesting questions about the ability of AUF1 isoforms to regulate the mRNA binding and decay-promoting activities of TTP and its family members as well as the ability of AUF1 proteins to serve as possible physical links between TTP and other mRNA decay proteins and structures.

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Figures

FIGURE 1.
FIGURE 1.
Interaction of AUF1p45 with hTTP and PABP. In this and subsequent figures, extracts were prepared in a lysis buffer from HEK 293 cells transfected with DNA encoding the HA- and FLAG-tagged expression constructs indicated at the top of each gel lane by the “+” sign. Total DNA transfected was 5.0 μg/100-mm dish of cells. For each Western blot shown, the immunoprecipitating antibody (IP) and the subsequent immunoblotting antibody (IB) are indicated to the left of each panel as are the positions of protein molecular mass standards. In some cases the blots are of WCL (50 μg of total protein per lane) to confirm expression of the respective proteins in the lysates before immunoprecipitation. HA-MARCKS served as a negative control as a non-TTP binding protein, and PABP served as a positive control as a known TTP binding protein (17) as indicated. As a further negative control, IP was performed using normal rabbit serum as indicated. The relevant immunoreactive protein species are indicated by the labeled arrows to the right of each blot. Each immunoprecipitation used 1 mg of cellular lysate protein as the starting material. See “Results” for additional details.
FIGURE 2.
FIGURE 2.
Interaction of hTTP with AUF1 isoforms and mutants. For A and B, abbreviations and other details are as described in the legend to Fig. 1. C shows a schematic representation of the domain structures of the four AUF1 isoforms as well as the related protein laAUF1. Each protein contains two non-identical RNA recognition motifs labeled as RRM1 and RRM2 and an eight amino acid glutamine-rich motif (Q) near the C terminus. The 19-amino acid region derived from exon 2 (X2) is shown for the p40 and p45 isoforms (dark gray box), whereas the 49-amino acid insert derived from exon 7 (X7), also known as the GY rich region, is shown for the p42 and p45 isoforms as well as for the laAUF1 protein. D, the various expression constructs that expressed variants of FLAG-AUF1p45 (WT) show the deletions of AUF1p45 as well as the FLAG-AUF1-X7 (49 amino acids) segment composing the GY motif. In all cases, these were transfected with HA-hTTP, as shown at the top of the panel. See “Results” for other details.
FIGURE 3.
FIGURE 3.
Interaction of the TZF domain of hTTP with AUF1p45. Abbreviations and other details are as described in the legend to Fig. 1. In A, FLAG-AUF1p45 was expressed with various TTP constructs, including the HA empty vector (lane 1), the full-length WT HA-hTTP, the full-length mutant TTP with a cysteine 124 to arginine mutation (C124R), and the HA-TZF domain alone from hTTP. In B, the possibility of direct protein-protein interactions was tested by incubation of recombinant GFP-TZF (hTTP) with purified recombinant His-AUF1p45 (B1 and B2). IP and IB were performed with the indicated antibodies. Each IP was conducted using 1 μg of each purified recombinant protein. See “Results” for further details.
FIGURE 4.
FIGURE 4.
Binding of other TTP family members to AUF1p45. Abbreviations and other details are as described in the legend to Fig. 1. FLAG-AUF1p45 was expressed with various TTP family member constructs. The expressed TTP-related proteins ZFP36L1 and ZFP36L2 were the human proteins, whereas the expressed ZFP36L3 was the mouse protein.
FIGURE 5.
FIGURE 5.
Effects of co-expression of AUF1p45 with hTTP on expression of a TNF-based RNA target. A Northern blot shows the effects of increasing amounts of transfected hTTP DNA on the stability of a TNF-based mRNA probe in the presence and absence of a constant concentration of co-transfected and expressed AUF1p45. Lanes 1 and 7 represent RNA samples from cells mock-transfected without DNA. In the other lanes, CMV.TNFα was transfected into HEK 293 cells in the presence of vector alone (BS+) (lane 2) or BS+ vector plus CMV.FLAG-AUF1p45 (lane 8). Lanes 3–6 and 9–12 show the effects of CMV.HA-hTTP DNA at the indicated concentrations of transfected DNA in the absence (lanes 3–6) and the presence (lanes 9–12) of a constant amount of transfected-expressed AUF1p45. In all cases vector DNA was added as needed to make the total amount of transfected DNA 5 μg/plate. The northern blots were probed with 32P-labeled probes for TNF, hTTP, AUF1p45, and GAPDH, as indicated to the left of the panels. Note the two species of the TNF-based RNA seen in the presence of TTP (double lines) in both panels. The position of the 18 S ribosomal RNA is indicated to the right of each panel. This figure is representative of three similar experiments.
FIGURE 6.
FIGURE 6.
Effects of AUF1p45 on hTTP binding to a TNF-based RNA probe. Cytosolic extracts of HEK 293 cells transfected with vector alone (BS) or vectors expressing HA-hTTP alone or Flag-AUF1p45 alone were used in RNA gel shift analyses using a 5′-biotin-labeled TNF ARE-based RNA probe. A shows RNA gel shifts using protein extracts/cell lysates containing identical amounts of empty vector-transfected cytosolic protein and decreasing amounts of FLAG-AUF1p45 incubated in the presence or absence of a constant concentration of FLAG-hTTP. The migration positions of the hTTP-ARE complexes and that of the RNA probe alone are indicated with arrows to the right of Fig. 6A1. 6A2 shows an immunoblot performed with 10 times the amount of cellular proteins as used in the gel shift analysis from the same cellular extracts (A1 and B1), demonstrating expression of the epitope-tagged proteins. B1 shows a gel shift analysis in which extracts containing a constant amount of cellular protein (1 μg) with or without FLAG-AUF1p45 were incubated with varying amounts of FLAG-hTTP-containing cell extracts (1.0 to 0 μg). The asterisks indicate endogenous HEK 293 cell proteins that could shift small amounts of probe in the absence of hTTP. Shown in B2 is the graphic representation of the results of four experiments identical to that shown in B1, shown as the means ± S.E. Asterisks indicate statistical differences between the means of each pair at p < 0.05. Below the histogram are shown the corresponding gel lanes in B1, and the amount of cell extracts of each type used in the gel shift experiments. See “Results” for further details. C1 shows a gel shift analysis in which FLAG-hTTP-containing HEK 293 cellular extracts in varying amounts (1.0–0 μg) with or without a constant amount of purified recombinant His-AUF1p45 (20 ng) were incubated together. The asterisks indicate endogenous HEK 293 cell proteins that could shift small amounts of probe in the absence of hTTP. The thick arrow on the left indicates the quantitated area of the shifted ARE-protein complex. C2 is the graphic representation of the results of three experiments identical to that shown in C1, shown as the means ± S.E. Asterisks indicate statistical differences between the means of each pair at p < 0.05. Below the histogram are shown the corresponding gel lanes in C1 and the amount of cell extracts of each type used in the gel shift experiments. Abbreviations and other details are as described in the legend to Fig. 1. NS, non-specific. See “Results” for further details.
FIGURE 7.
FIGURE 7.
Enhancement of TTP TZF:RNA complex formation by AUF1p45. A, shown is a schematic representation of the FRET-based GFP-TTP-TZF·RNA binding assay. Upon excitation at 485 nm, recombinant GFP-TTP-TZF emits with λmax = 511 nm. However, when bound to a Cy5-labeled RNA target, some energy from the excited GFP-TTP TZF protein may be transferred to the RNA-linked Cy5 moiety by FRET, resulting in diminution of quantum emission from GFP but enhanced emission from Cy5 (λmax = 665 nm). B, shown are fluorescence emission scans (λex = 485 nm) of binding reactions containing GFP-TTP-TZF (1 nm) and 0 (black), 0.5 (magenta), 1 (orange), 3 (green), and 20 nm (red) final concentrations of the Cy5-ARE11 RNA substrate. Each spectrum was corrected for Cy5 emission resulting from direct excitation at 485 nm by subtracting spectra from parallel samples lacking the GFP-TTP-TZF protein. C, shown are fluorescence emission scans (λex = 485 nm) of samples containing GPF-TTP-TZF (1 nm) and 0 (black solid line) or 20 nm (red dashed line) of an unlabeled ARE13 substrate. D, shown are representative plots of FRET efficiency (EFRET) from GFP-TTP-TZF as a function of Cy5-ARE11 RNA substrate concentration in reactions lacking AUF1 proteins (black circles) or containing 10 nm His-AUF1p45 (open circles) or 10 nm His-AUF1p37 (open triangles). Non-linear regression was performed as described under “Materials and Methods,” and resolved binding constants are listed in “Results.”
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References

    1. Taylor G. A., Carballo E., Lee D. M., Lai W. S., Thompson M. J., Patel D. D., Schenkman D. I., Gilkeson G. S., Broxmeyer H. E., Haynes B. F., Blackshear P. J. (1996) A pathogenetic role for TNF-α in the syndrome of cachexia, arthritis, and autoimmunity resulting from tristetraprolin (TTP) deficiency. Immunity 4, 445–454 - PubMed
    1. Carballo E., Blackshear P. J. (2001) Roles of tumor necrosis factor-α receptor subtypes in the pathogenesis of the tristetraprolin-deficiency syndrome. Blood 98, 2389–2395 - PubMed
    1. Carballo E., Gilkeson G. S., Blackshear P. J. (1997) Bone marrow transplantation reproduces the tristetraprolin deficiency syndrome in recombination activating gene-2−/− mice. Evidence that monocyte/macrophage progenitors may be responsible for TNFα overproduction. J. Clin. Invest. 100, 986–995 - PMC - PubMed
    1. Carballo E., Lai W. S., Blackshear P. J. (1998) Feedback inhibition of macrophage tumor necrosis factor-α production by tristetraprolin. Science 281, 1001–1005 - PubMed
    1. Lai W. S., Carballo E., Strum J. R., Kennington E. A., Phillips R. S., Blackshear P. J. (1999) Evidence that tristetraprolin binds to AU-rich elements and promotes the deadenylation and destabilization of tumor necrosis factor α mRNA. Mol. Cell. Biol. 19, 4311–4323 - PMC - PubMed

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