Selective degradation of AU-rich mRNAs promoted by the p37 AUF1 protein isoform

Abstract

An AU-rich element (ARE) consisting of repeated canonical AUUUA motifs confers rapid degradation to many cytokine mRNAs when present in the 3' untranslated region. Destabilization of mRNAs with AREs (ARE-mRNAs) is consistent with the interaction of ARE-binding proteins such as tristetraprolin and the four AUF1 isoforms. However, the association of the AUF1-mRNA interaction with decreased ARE-mRNA stability is correlative and has not been directly tested. We therefore determined whether overexpression of AUF1 isoforms promotes ARE-mRNA destabilization and whether AUF1 isoforms are limiting components for ARE-mRNA decay. We show that the p37 AUF1 isoform and, to a lesser extent, the p40 isoform possess ARE-mRNA-destabilizing activity when overexpressed. Surprisingly, overexpressed p37 AUF1 also destabilized reporter mRNAs containing a noncanonical but AU-rich 3' untranslated region. Since overexpressed p37 AUF1 could interact in vivo with the AU-rich reporter mRNA, AUF1 may be involved in rapid turnover of mRNAs that lack canonical AREs. Moreover, overexpression of p37 AUF1 restored the ability of cells to rapidly degrade ARE-mRNAs when that ability was saturated and inhibited by overexpression of ARE-mRNAs. Finally, activation of ARE-mRNA decay often involves a translation-dependent step, which was eliminated by overexpression of p37 AUF1. These data indicate that the p37 AUF1 isoform and, to some extent, the p40 isoform are limiting factors that facilitate rapid decay of AU-rich mRNAs.

FIG. 1.

Steady-state reporter mRNA analysis with overexpression of Flag-AUF1 proteins. (A) ARE sequences cloned into the 3′ UTR of β-Gal reporter plasmids, depicted as RNA nucleotides with the canonical AUUUA motifs underlined. AU, wild-type ARE from the GM-CSF 3′ UTR; GC, AU sequence with G and C point mutations. Also shown is the 3′ UTR of the control plasmid pEGFP-C3. (B) CHO cells were cotransfected with 1.0 μg of either the Flag expression plasmid or each individual Flag-AUF1 isoform per 10⁷ cells and plasmids encoding mRNAs containing the GM-CSF ARE (AU) or the GC mutant ARE (GC). Cells were transfected with 0.5 μg of a GFP expression vector that lacks a significant AU-rich 3′ UTR. At 24 h posttransfection, total RNA was isolated, and equal amounts were analyzed by Northern blot hybridization using probes prepared from the lacZ gene. A representative blot is shown. Autoradiograms were quantified by densitometry, and the ratio of the β-Gal/GFP mRNAs for the vector Flag sample (lane 1) was set to 100%. The ratio of normalized β-Gal to GFP mRNA indicates the percent abundance of β-Gal mRNA relative to that of the untreated vector sample.

FIG. 2.

mRNA stability analysis with Flag-AUF1 proteins. CHO cells were transfected as described in Fig. 1 and then treated with 5 μg of actinomycin D/ml 24 h posttransfection for the indicated time periods prior to isolation of total RNA and Northern blot analysis. Representative blots are shown. Insets represent overexposures of β-Gal mRNA samples cotransfected with AUF1 proteins to permit better visualization of the low-abundance mRNA.

FIG. 3.

Analysis of the effect of p37 (A) and p40 (B) AUF1 supplementation on mRNA half-life. CHO cells were cotransfected with Flag-p37 or Flag-p40 and the vector alone and reporters containing the GM-CSF ARE (AU) or the GC mutant ARE (GC). The β-Gal mRNA level was normalized to the GFP mRNA level determined by densitometry of the results from Fig. 2 to correct for transfection efficiency. Normalized β-Gal mRNA decay curves were plotted as percentages of the initial mRNA concentration versus time of actinomycin D chase. Half-lives (t_1/2) of GC-mRNA (with and without p40 AUF1) were estimated by extrapolation of decay curves.

FIG. 4.

Overexpressed p37 AUF1 is associated with AU-rich mRNAs in vivo. 293 cells were transfected with 1.0 μg of plasmids expressing β-Gal mRNAs and an ARE or GC reporter mRNA, 0.5 μg of a plasmid expressing Flag alone, or 0.5 or 2.0 μg of a plasmid expressing Flag-p37 AUF1. eIF4G and Flag were immunoprecipitated from cell lysates normalized to equal amounts of β-Gal mRNAs, and β-Gal mRNA-specific RT-PCR was carried out. The products of RT-PCR were resolved by agarose gel electrophoresis and visualized by staining with ethidium bromide.

FIG. 5.

p37 AUF1 relieves saturation of the ARE-mRNA decay pathway. (A) CHO cells were cotransfected with either 1.0 μg of plasmid vector or the Flag-p37 plasmid and 1.5 or 4.5 μg of the β-Gal ARE or GC control reporter plasmids per 10⁷ cells. Total RNA was isolated 24 h posttransfection, and Northern analysis was performed. (B) CHO cells were transfected as in panel A but with a fixed amount of the reporter plasmid (1.5 μg) and an increasing amount of Flag-p37 plasmid DNA (0.1, 0.5, and 1.0 μg), as shown. A representative blot is shown. Autoradiograms were quantified by densitometry, and data were normalized by transfection efficiency, as determined by GFP β-Gal control mRNA with the vector alone set at 100%, as described in the legend to Fig. 1.

FIG. 6.

Flag-p37 expression inhibits cycloheximide-mediated stabilization of ARE-mRNA decay. (A) CHO cells were cotransfected with Flag-p37 AUF1 or vector alone, along with β-Gal reporter plasmid DNAs. At 24 h posttransfection, cells were treated with 100 μg of cycloheximide (CHX)/ml for 4 h prior to isolation of total RNA and Northern blot analysis. Autoradiograms were quantified by densitometry, β-Gal mRNA levels were normalized to the GFP control mRNA, and levels were expressed relative to that of the untreated and untransfected control GC-mRNA, which was set at 100%. (B) Untransfected (left) and Flag-p37-transfected (right) CHO cells were treated with CHX and then used to prepare whole-cell lysates. Equal amounts of total protein were resolved by SDS-PAGE and immunoblotted for endogenous AUF1 or Flag. The blot was stripped and reprobed with an antibody specific for tubulin or GFP to verify equal loading. Representative blots are shown.

FIG. 7.

Destabilization of ARE-mRNA by Flag-p37 in various cell lines. (A) The indicated cell lines were cotransfected with vector alone or the indicated amounts of Flag-p37, along with the β-Gal and GFP reporter plasmids. At 24 h posttransfection, total RNA was isolated and Northern blot analysis was performed. (B) Equal amounts of whole-cell lysates prepared from the various cell lines were resolved by SDS-PAGE and immunoblotted for endogenous or Flag-tagged AUF1 proteins. Representative blots from at least three independent experiments are shown. Autoradiograms were quantified by densitometry, β-Gal mRNA levels were normalized to the GFP control mRNA, and levels were expressed relative to that of the untreated and untransfected control GC-mRNA, which set at 100%.