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PMID:12198173

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Citation

Stoecklin, G, Colombi, M, Raineri, I, Leuenberger, S, Mallaun, M, Schmidlin, M, Gross, B, Lu, M, Kitamura, T and Moroni, C (2002) Functional cloning of BRF1, a regulator of ARE-dependent mRNA turnover. EMBO J. 21:4709-18

Abstract

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.

Links

PubMed PMC126184

Keywords

3' Untranslated Regions/genetics; 3T3 Cells; Animals; Butyrate Response Factor 1; Cloning, Molecular; Codon, Nonsense; Cytokines/genetics; DNA, Complementary/genetics; DNA-Binding Proteins; Fibrosarcoma/chemistry; Fibrosarcoma/pathology; Frameshift Mutation; Genes, Reporter; Genetic Complementation Test; Humans; Immediate-Early Proteins/analysis; Immediate-Early Proteins/chemistry; Mice; 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; 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 Fingers/genetics

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

HUMAN:TTP

located_in

GO:0005634: nucleus

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

HUMAN:TTP

located_in

GO:0005737: cytoplasm

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

HUMAN:TISB

located_in

GO:0005634: nucleus

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

HUMAN:TISB

located_in

GO:0005737: cytoplasm

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

HUMAN:TISB

enables

GO:0035925: mRNA 3'-UTR AU-rich region binding

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

HUMAN:TISB

involved_in

GO:0061158: 3'-UTR-mediated mRNA destabilization

ECO:0000315: mutant phenotype evidence used in manual assertion

P

Seeded From UniProt

complete

HUMAN:TISB

involved_in

GO:0043488: regulation of mRNA stability

ECO:0000315: mutant phenotype evidence used in manual assertion

P

Seeded From UniProt

complete

HUMAN:TTP

part_of

GO:0005737: cytoplasm

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

HUMAN:TTP

part_of

GO:0005634: nucleus

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete


See also

References

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