No abstract

Many labile mammalian mRNAs are targeted for rapid cytoplasmic turnover by the presence of A + U-rich elements (AREs) within their 3'-untranslated regions. These elements are selectively recognized by AUF1, a component of a multisubunit complex that may participate in the initiation of mRNA decay. In this study, we have investigated the recognition of AREs by AUF1 in vitro using oligoribonucleotide substrates. Gel mobility shift assays demonstrated that U-rich RNA targets were specifically bound by AUF1, generating two distinct RNA-protein complexes in a concentration-dependent manner. Chemical cross-linking revealed the interaction of AUF1 dimers to form tetrameric structures involving protein-protein interactions in the presence of high affinity RNA targets. From these data, a model of AUF1 association with AREs involving sequential dimer binding was developed. Using fluorescent RNA substrates, binding parameters of AUF1 dimer-ARE and tetramer-ARE equilibria were evaluated in solution by fluorescence anisotropy measurements. Using two AUF1 deletion mutants, sequences C-terminal to the RNA recognition motifs are shown to contribute to the formation of the AUF1 tetramer.ARE complex but are not obligate for RNA binding activity. Kinetic studies demonstrated rapid turnover of AUF1.ARE complexes in solution, suggesting that these interactions are very dynamic in character. Taken together, these data support a model where ARE-dependent oligomerization of AUF1 may function to nucleate the formation of a trans-acting, RNA-destabilizing complex in vivo.

Proteins binding A ؉ U-rich elements (AREs) contribute to the rapid cytoplasmic turnover of mRNAs containing these sequences. However, this process is a regulated event and may be accelerated or inhibited by myriad signal transduction systems. For example, monocyte adherence at sites of inflammation or tissue injury is associated with inhibition of ARE-directed mRNA decay, which contributes to rapid increases in cytokine and inflammatory mediator production. Here, we show that acute exposure of THP-1 monocytic leukemia cells to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate mimics several features of monocyte adherence, including rapid induction and stabilization of ARE-containing mRNAs encoding interleukin-1␤ and tumor necrosis factor ␣. Additionally, TPA treatment alters the activity of cytoplasmic complexes that bind AREs, including complexes containing the ARE-specific, mRNA-destabilizing factor, AUF1. Analyses of AUF1 from control and TPA-treated cells indicated that posttranslational modifications of the major cytoplasmic isoform, p40 AUF1 , are altered concomitant with changes in RNA binding activity and stabilization of ARE-containing mRNAs. In particular, p40 AUF1 recovered from polysomes was phosphorylated on Ser 83 and Ser 87 in untreated cells but lost these modifications following TPA treatment. We propose that selected signal transduction pathways may regulate ARE-directed mRNA turnover by reversible phosphorylation of polysome-associated p40 AUF1 .In eukaryotes, cytoplasmic mRNA stability is an important checkpoint in the control of gene expression. Many mRNAs encoding regulatory proteins like cytokines, inflammatory mediators, and oncoproteins are constitutively unstable. This ensures that the steady-state levels of these mRNAs, and hence their potential for translation, remain low but also that new steady-state levels are approached quickly following changes in the rate of mRNA synthesis (reviewed in Ref. 1). In mammals, a common feature of many unstable mRNAs is the presence of an A ϩ U-rich element (ARE) 1 within the 3Ј-untranslated region (3Ј-UTR). These elements range from 40 to 150 nucleotides in length and exhibit significant variability in sequence composition, but they usually include one or more AUUUA motifs within a U-rich context (2). In general, mRNA turnover mediated by AREs consists of rapid 3Ј 3 5Ј shortening of the poly(A) tail, followed by decay of the mRNA body (3, 4).The regulation of mRNA decay kinetics by AREs involves their association with any of a number of cellular ARE-binding factors (reviewed in Ref. 5). One such factor, AUF1 (also referred to as heterogeneous nuclear ribonucleoprotein D), is expressed as a family of four protein isoforms resulting from alternative splicing of a common pre-mRNA (6). The larger isoforms, designated by their apparent molecular weights as p42 AUF1 and p45 AUF1 , are largely nuclear (7), probably due to the presence of a binding determinant for components of the nuclear scaffold (8). By contrast, p37 AUF1 and p40 AUF1 lack this seq...

AUF1 is an RNA-binding protein that contains two nonidentical RNA recognition motifs (RRMs). AUF1 binds to A ؉ U-rich elements (AREs) with high affinity. The binding of AUF1 to AREs is believed to serve as a signal to an mRNA-processing pathway that degrades mRNAs encoding many cytokines, oncoproteins, and G protein-coupled receptors. Because the ARE binding activity of AUF1 appears central to the regulation of many important genes, we analyzed the domains of the protein that are important for this activity. Examination of the RNA binding affinity of various AUF1 mutants suggests that both RRMs may be required for binding to the human c-fos ARE. However, the two RRMs together are not sufficient. Highest affinity binding of AUF1 to an ARE requires an alanine-rich region of the N terminus and a short glutamine-rich region in the C terminus. In addition, the N terminus is required for dimerization of AUF1. However, AUF1 binds an ARE as a hexameric protein. Thus, protein-protein interactions are important for high affinity ARE binding activity of AUF1.RNA processing is an important component of regulated gene expression in eukaryotic cells. Together, the rates of transcription, pre-mRNA splicing, mRNA transport, translation, and mRNA degradation determine the steady-state amount of mRNA, and hence protein, that will be present in a cell at a given time. Each of these processes of RNA metabolism involves RNA-binding proteins that exhibit specific protein-RNA interactions (reviewed in Ref. 1). Thus, defining how such proteins interact with their RNA substrates is integral to understanding the complex control of RNA processing.A variety of conserved protein motifs mediate specific protein-RNA interactions (2). Perhaps the most common and well characterized of these motifs is the RNA recognition motif (RRM), 1 also called a consensus sequence RNA-binding domain. This motif consists of 80 -90 amino acids containing two conserved sequences: a highly conserved octamer motif, RNP-1, and a less conserved hexamer motif, RNP-2 (2, 3). The general structure of an RRM consists of a ␤␣␤-␤␣␤ folding pattern in a four-stranded ␤-sheet with the two ␣-helices packed against one face of the sheet. The RNP-1 and RNP-2 motifs lie on the two central strands at the center of the ␤-sheet. These motifs probably provide general RNA binding activity. The mechanisms by which RRMs provide sequence-specific or structurespecific RNA recognition are unknown. However, recognition of specific targets is thought to be provided by unique amino acids located in intradomain loops and tails. Nonetheless, this seemingly simple view is complicated by two observations. (i) Multiple RRMs within some proteins are required for high affinity RNA binding (see Ref. 3 and references therein); and (ii) complex communication can occur between amino acids in the intradomain loops and tails (4). Thus, the efforts of many laboratories are being directed toward dissecting the molecular mechanisms of specific RNA recognition by RNA-binding proteins.We previously molecul...