Abstract
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.
Highlights
RNA processing is an important component of regulated gene expression in eukaryotic cells
This seemingly simple view is complicated by two observations. (i) Multiple RNA recognition motifs (RRMs) within some proteins are required for high affinity RNA binding; and (ii) complex communication can occur between amino acids in the intradomain loops and tails (4)
Identification of the Domains of AUF1 Critical for Its High Affinity A ؉ U-rich elements (AREs) Binding Activity—Using quantitative electrophoretic mobility shift assays, we showed previously that purified, recombinant His6-p37AUF1 fusion protein binds the human c-fos ARE with an apparent Kd of 7.8 Ϯ 0.4 nM. (The wild-type fusion protein will hereafter be referred to as His6AUF1-(1–286).) As shown in Fig. 1, the predicted AUF1 polypeptide contains two tandem, nonidentical RRMs
Summary
RNA processing is an important component of regulated gene expression in eukaryotic cells. The rates of transcription, pre-mRNA splicing, mRNA transport, translation, and mRNA degradation determine the steady-state amount of mRNA, and 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). 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. Recognition of specific targets is thought to be provided by unique amino acids located in intradomain loops and tails. (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). Additional protein-protein interactions are involved in the high affinity ARE binding activity of AUF1
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