Structural Determinants in AUF1 Required for High Affinity Binding to A + U-rich Elements

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.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...

TcUBP-1, an mRNA Destabilizing Factor from Trypanosomes, Homodimerizes and Interacts with Novel AU-rich Element- and Poly(A)-binding Proteins Forming a Ribonucleoprotein Complex

“…Moreover, these residues were involved in structure stability of the free protein (23). In heterogeneous nuclear RNP-D, the C-terminal of RRM2 was important in conferring high affinity for ARE binding (24). This phenomenon suggests that those residues A, GST pull-down assay between GST or GST-TcPABP1 and TcUBP-1 or TcUBP-2 proteins, in the absence of RNA.…”

Heterogeneous nuclear ribonucleoprotein D0 contains transactivator and DNA-binding domains

“…Its role in regulating the half-life of mRNA species containing an AU-rich element is well characterized [3,4]. Although it is more abundant in the nucleus than in the cytoplasm [30], no nuclear function has been assigned to hnRNP D0.…”

“…The human and murine proteins are 97 % identical, suggesting an evolutionarily conserved function for hnRNP D0 [2]. hnRNP D0 has been implicated in the regulation of mRNA stability by recognizing AU-rich elements present in the 3h untranslated region of several mRNA species [3,4], and also binds the telomeric repeat and the 3h splice site sequence [5]. The B isoform of hnRNP D0 recognizes a motif in the promoter region of the human complement receptor 2 (CR2) gene [6] and co-precipitates with the TATAbinding protein (TBP), suggesting that it may represent a transcription factor [7].…”

“…Only one RBD of hnRNP D0 is required for the specific recognition of UUAG RNA sequences, and the sequence preference is modified by the presence of the 19-amino-acid insertion [12]. The binding of the shortest isoform of hnRNP D0, which lacks both alternative exons, to the AU-rich element of c-fos mRNA requires both RBDs, as well as parts of the C-and N-termini for highest affinity [3]. Although these studies have provided some information on the mechanism of RNA binding of hnRNP D0, there is no information on its DNA-binding characteristics.…”

Developmental expression of AUF1 and HuR, two c-myc mRNA binding proteins

“…Since the alternative exons can change the speci®city and/or a nity of AUF1 isoforms for AREs (Kajita et al, 1995;Wagner et al, 1997), it was important to determine whether the respective mRNAs showed identical or distinct patterns of expression. For this purpose, we designed speci®c probes for each AUF1 mRNA category (see Materials and methods and Figure 4a) and used them in S1 nuclease analysis.…”