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

Control of mRNA stability is critical for expression of short-lived transcr ipts from cytokines and protooncogenes. Regulation involves an AU-rich elem ent (ARE) in the 3' untranslated region (3'UTR) and cognate trans-acting fa ctors thought to promote either degradation or stabilization of the mRNA. I n this study we present a novel approach using somatic cell genetics design ed to identify regulators of interleukin-3 (IL-3) mRNA turnover. Mutant cel l lines were generated from diploid HT1080 cells transfected with a reporte r 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. Follo wing chemical mutagenesis and selection of GFP-overexpressing cells, we cou ld isolate three mutant clones (slowA, slowB, and slowC) with a specific, t rans-acting defect in IL-3 mRNA degradation, while the stability of IL-2 an d tumor necrosis factor alpha reporter transcripts was not affected. Somati c cell fusion experiments revealed that the mutants are genetically recessi ve and form two complementation groups. Expression of the tristetraprolin g ene in both groups led to reversion of the mutant phenotype, thereby linkin g this gene to the IL-3 mRNA degradation pathway. The genetic approach desc ribed here should allow identification of the defective functions by gene t ransfer and is also applicable to the study of other mRNA turnover pathways .