Somatic mRNA turnover mutants implicate tristetraprolin in the interleukin-3 mRNA degradation pathway

Abstract

Control of mRNA stability is critical for expression of short-lived transcripts from cytokines and proto-oncogenes. Regulation involves an AU-rich element (ARE) in the 3' untranslated region (3'UTR) and cognate trans-acting factors thought to promote either degradation or stabilization of the mRNA. In this study we present a novel approach using somatic cell genetics designed to identify regulators of interleukin-3 (IL-3) mRNA turnover. Mutant cell lines were generated from diploid HT1080 cells transfected with a reporter construct containing green fluorescent protein (GFP) linked to the IL-3 3'UTR. GFP was expressed at low levels due to rapid decay of the mRNA. Following chemical mutagenesis and selection of GFP-overexpressing cells, we could isolate three mutant clones (slowA, slowB, and slowC) with a specific, trans-acting defect in IL-3 mRNA degradation, while the stability of IL-2 and tumor necrosis factor alpha reporter transcripts was not affected. Somatic cell fusion experiments revealed that the mutants are genetically recessive and form two complementation groups. Expression of the tristetraprolin gene in both groups led to reversion of the mutant phenotype, thereby linking this gene to the IL-3 mRNA degradation pathway. The genetic approach described here should allow identification of the defective functions by gene transfer and is also applicable to the study of other mRNA turnover pathways.

FIG. 1

(A) Schematic representation of the reporter constructs GFPIL3-wt and -3a. GFP is flanked 5′ by a 1.9-kb fragment of the IL-3 promoter and 3′ by an XbaI-SpeI fragment of murine IL-3 containing part of exon 5 including the entire 3′UTR and the poly(A) site. Dark shaded box, GFP, light shaded boxes, IL-3 cDNA sequences. (B) Nucleotide sequence of the core AUUUA motif cluster within the ARE of IL-3, which contains 6 partially overlapping AUUUA pentamers. In mutation 3a, three pentamers were mutated to AGGUA, as indicated by double asterisks. This is the minimal mutation that abrogates the destabilizing effect of the ARE (41).

FIG. 2

Expression of the GFP reporter construct in the parental cell line HT-GFPIL3-wt (A), the control mutant HT-GFPIL3-3a (B), and the mutants 12-2 (C), slowA (D), slowB (E), and slowC (F). (Left) Analysis of GFP expression by flow cytometry. (Center) Analysis of reporter mRNA stability by actinomycin D chase experiments and Northern blotting. RNA was isolated 0, 1, 2, and 3 h after inhibition of transcription with 5 μg of actinomycin D/ml. Twenty-five micrograms of total RNA was resolved on 1.1% agarose-formaldehyde gels and transferred onto Hybond N+ membranes, and GFPIL3 reporter mRNA was detected with a radiolabeled SP6 probe against the IL-3 3′UTR. All blots were stripped and rehybridized with a β-actin probe as a loading control. (Right) Signal intensities were quantified using a PhosphorImager and ImageQuant software. Values of GFPIL3 were normalized to actin and plotted as relative mRNA levels against time. mRNA half-lives were calculated by means of linear regression and are summarized in Table 2.

FIG. 3

Decay of mRNA from stably transfected IL-3, IL-2, and TNF-α reporter constructs in control and mutant cell lines. Actinomycin D chase experiments and Northern blot analysis were performed as described for Fig. 2. For quantification (right), signal intensities were normalized to that of actin and plotted as percentages of the initial value against time. (A) Cells were transfected with HindIL3neo, a 5.3-kb genomic IL-3 construct that uses the IL-3 promoter. (B) Expression of MXIL3neo, where transcription of IL-3 is driven by an MMLV LTR promoter. For panels A and B, a HindIII-XbaI fragment of the IL-3 coding region was used to generate a radiolabeled SP6 RNA probe that specifically recognizes IL-3 mRNA but not GFPIL3 mRNA, which is also expressed in the mutants. (C) Cells were transfected with neoMXβglobin-IL2, a plasmid that contains a β-globin reporter gene linked to the entire 3′UTR of murine IL-2. (D) Expression of neoMXβglobin-TNF-α, containing the 3′UTR of murine TNF-α. In panels C and D, an SP6 probe against β-globin was used to detect reporter mRNA.

FIG. 4

Decay of GFPIL3 mRNA in hybrid cell lines. (A) Somatic cell fusion was performed between parental HT1080 cells expressing pBABEpuro and the hygromycin-resistant trans mutants slowA, slowB, and slowC, as well as the cis mutant HT-GFPIL3-3a. After selection with puromycin and hygromycin, hybrids were analyzed by actinomycin D chase experiments and Northern blotting, as described for Fig. 2. (B) Intermutant hybrids were generated by crossing mutants slowA, slowB, and slowC with each other (as specified in Materials and Methods). GFPIL3 reporter mRNA, expressed by both fusion partners, was detected with an SP6 probe directed against the GFP coding region in order to prevent hybridization with IL-3 mRNA from the HindIL3neo-transfected fusion partner.

FIG. 5

Transfection of tristetraprolin (mTTP.tag) into slowA and slowC, and analysis of GFP reporter gene expression in subclones. (A) Western blot analysis with untransfected slowA (lane 1), transfected slowA-TTP (lane 2), and subclones of slowA-TTP (lanes 3 to 6). Similarly, mTTP.tag expression was analyzed in untransfected slowC (lane 7), transfected slowC-TTP (lane 8), and subclones of slowC-TTP (lanes 9 to 11). A lysate of 1 × 10⁶ to 2 × 10⁶ cells was resolved on a 12% polyacrylamide-SDS gel and blotted onto an Immobilon-P membrane, and the anti-myc antibody 9E10 served to detect the myc- and His-tagged protein, which has an approximate molecular weight of 46 kDa. GFP reporter gene expression was analyzed in the following subclones: A-TTP-1 (B), A-TTP-13 (C), A-TTP-15 (D), C-TTP-4 (E), C-TTP-10 (F), and C-TTP-18 (G). GFP levels were assayed by flow cytometry (left panel), and reporter mRNA stability was measured by actinomycin D chase experiments (right panel). Northern blot analysis was performed as described for Fig. 2.

FIG. 6

Northern blot analysis of endogenous TTP (hTTP) expression in parental HT-GFPIL3-wt cells and mutants slowA and slowC. Cells were grown in low concentrations of serum (0.5% FCS) for 24 h, and RNA was isolated before (lanes 1 to 3) or after stimulation with TPA (10 ng/ml) for 45 min (lanes 4 to 6). Fifty micrograms of total RNA was used for each lane, and the blot was hybridized with a random-primed probe generated from human TTP cDNA. The membrane was stripped and rehybridized with a β-actin probe as a loading control.