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PMID: 10805719 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

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

Molecular and cellular biology ·Vol. 20 ·No. 11 ·2000-06-00 ·Pages 3753-63

Stoecklin G, Ming XF, Looser R, Moroni C

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.

MeSH Terms
Animals DNA-Binding Proteins Genes, Reporter Genetic Complementation Test Humans Immediate-Early Proteins Interleukin-3/genetics Mice Mutagenesis Phenotype Proteins/genetics,metabolism RNA Stability/genetics RNA, Messenger/metabolism Tristetraprolin Tumor Cells, Cultured Zinc Fingers
Chemicals
DNA-Binding Proteins Immediate-Early Proteins Interleukin-3 Proteins RNA, Messenger Tristetraprolin ZFP36 protein, human Zfp36 protein, mouse
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Stoecklin G
Institute of Medical Microbiology, University of Basel, CH-4003 Basel, Switzerland.
Ming X F
Looser R
Moroni C
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Article Info
Journal
Molecular and cellular biology
Abbr.
Mol Cell Biol
ISSN
0270-7306
DOI
10.1128/MCB.20.11.3753-3763.2000
Published
2000-06-00
Pages
3753-63
Language
English
Region
United States
NLM ID
8109087
PMCID
PMC85689
Subset
IM
External Links
PubMed source DOI: 10.1128/MCB.20.11.3753-3763.2000 PMC Full Text View journal details
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