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

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.

Concurrent versus individual binding of HuR and AUF1 to common labile target mRNAs

“…This scheme complements a popular displacement model whereby decay-promoting and stability-promoting RBPs are thought to compete for the same binding site (Park et al, 2000;Ming et al, 2001;Stoecklin et al, 2002;Cok et al, 2004). The general association of RBPs to common target RNAs was seen predominantly in the nucleus, but was very reduced in the cytoplasm.…”

“…According to a widely established view, HuR/ELAV proteins can compete with AUF1 (Park et al, 2000;Cok et al, 2004) or with other decay-promoting RBPs (Ming et al, 2001;Stoecklin et al, 2002) for binding to the same ARE on specific target mRNAs, but other regulatory schemes have not been formally tested. Moreover, no studies have directly assessed the endogenous association of HuR and AUF1 with target mRNAs.…”

A novel mechanism of tumor suppression by destabilizing AU-rich growth factor mRNA

“…As with classical loss-of-function tumor suppressor genes (Hanahan and Weinberg, 2000;Knudson, 2000), TTP or a closely related gene may be mutated in the tumor cells and transfection restores the missing function. We have sequenced the entire coding region of TTP and BRF1, a structurally related AREbinding zinc-finger protein that also regulates cytokine mRNA turnover (Lai et al, 2000;Stoecklin et al, 2002). In V2D1 tumor cells, no mutation was detected in either gene, and transcripts were of expected size and expressed at the same levels as in parental PB-3c cells (data not shown).…”

“…The mechanism of mRNA stabilization is likely to involve ARE-binding proteins as adaptors between the signalling cascade and the decay machinery (Carballo et al, 2001;Mahtani et al, 2001). ARE-binding proteins with assigned functions in vivo include TTP, butyrate response factor-1 (BRF1) and AUF1, all of which promote degradation, as well as HuR that exerts an mRNA stabilizing effect (Carballo et al, 1998;Fan and Steitz, 1998;Peng et al, 1998;Loflin et al, 1999;Lai et al, 2000;Stoecklin et al, 2002).…”

Post‐transcriptional regulation of gene expression by degradation of messenger RNAs

“…More recent studies have identified other factors capable of binding AREs with various degrees of avidity. These include selected nuclear ribonucleic proteins (hnRNP A1, hnRNP C (Hamilton et al, 1993), hnRNP D, also known as AUF-1 (Brewer, 1991)), RNA-binding proteins displaying enzymatic activities (GAPDH (Nagy and Rigby, 1995), AUH (Nakagawa et al, 1995)), other HuR-like proteins, belonging to the ELAV family (embryonic lethal abnormal vision) and related to the Drosophila neuron-specific RNA-binding proteins (Hel-N1, HuC, HuD, (Levine et al, 1993;Good, 1995;Ma et al, 1996) and HuR (Fan and Steitz, 1998;Peng et al, 1998), heat shock protein 70 (Hsp70) and others (AUBP (Malter and Hong, 1991), AU-A, AU-B, AU-C (Bohjanen et al, 1991(Bohjanen et al, , 1992, tristetraprolin (Taylor et al, 1996b), butyrate response factor-1 (BRF1), encoding a zinc finger protein homologous to tristetraprolin (Stoecklin et al, 2002), KSRP ), TIA-1 and TIAR (Piecyk et al, 2000)). …”