Functional cloning of BRF1, a regulator of ARE-dependent mRNA turnover

• Published in: The EMBO journal 2002-09, Vol.21 (17), p.4709-4718
• Main Authors: Stoecklin, Georg, Colombi, Marco, Raineri, Ines, Leuenberger, Sabrina, Mallaun, Michel, Schmidlin, Martin, Gross, Brigitte, Lu, Min, Kitamura, Toshio, Moroni, Christoph
• Format: Article
• Language: English
• Subjects: 3' Untranslated Regions - genetics; 3T3 Cells; Animals; AU-rich element; Butyrate Response Factor 1; Cloning; Cloning, Molecular; Codon, Nonsense; Cytokines - genetics; Decay; DNA, Complementary - genetics; DNA-Binding Proteins; EMBO36; ERF-1; Fibrosarcoma - chemistry; Fibrosarcoma - pathology; Frameshift Mutation; Genes, Reporter; Genetic Complementation Test; Humans; Immediate-Early Proteins - analysis; Immediate-Early Proteins - chemistry; Mice; mRNA stability; Mutation; Neoplasm Proteins - genetics; Neoplasm Proteins - isolation & purification; Phosphatidylinositol 3-Kinases - antagonists & inhibitors; RNA Stability; RNA, Messenger - metabolism; RNA, Small Interfering; RNA, Untranslated - metabolism; Saccharomyces cerevisiae Proteins; siRNA; Structure-Activity Relationship; Subcellular Fractions - chemistry; TATA-Binding Protein Associated Factors; Transcription Factor TFIIIB; Transcription Factors - genetics; Transcription Factors - isolation & purification; Transcription Factors - physiology; Transfection; Tristetraprolin; Tumor Cells, Cultured - chemistry; zinc finger protein; Zinc Fingers - genetics
• ISSN: 0261-4189; 1460-2075; 1460-2075
• DOI: 10.1093/emboj/cdf444

To identify regulators of AU‐rich element (ARE)‐dependent mRNA turnover we have followed a genetic approach using a mutagenized cell line (slowC) that fails to degrade cytokine mRNA. Accordingly, a GFP reporter construct whose mRNA is under control of the ARE from interleukin‐3 gives an increased fluorescence signal in slowC. Here we describe rescue of slowC by a retroviral cDNA library. Flow cytometry allowed us to isolate revertants with reconstituted rapid mRNA decay. The cDNA was identified as butyrate response factor‐1 (BRF1), encoding a zinc finger protein homologous to tristetraprolin. Mutant slowC carries frame‐shift mutations in both BRF1 alleles, whereas slowB with intermediate decay kinetics is heterozygous. By use of small interfering (si)RNA, independent evidence for an active role of BRF1 in mRNA degradation was obtained. In transiently transfected NIH 3T3 cells, BRF1 accelerated mRNA decay and antagonized the stabilizing effect of PI3‐kinase, while mutation of the zinc fingers abolished both function and ARE‐binding activity. This approach, which identified BRF1 as an essential regulator of ARE‐dependent mRNA decay, should also be applicable to other cis ‐elements of mRNA turnover.