Abstract
AU-rich element binding/degradation factor 1 (AUF1) plays a role in destabilizing mRNAs by forming complexes with AU-rich elements (ARE) in the 3′-untranslated regions. Multiple AUF1-ARE complexes regulate the translation of encoded products related to the cell cycle, apoptosis, and inflammation. AUF1 contains two tandem RNA recognition motifs (RRM) and a Gln- (Q-) rich domain in their C-terminal region. To observe how the two RRMs are involved in recognizing ARE, we obtained the AUF1-p37 protein covering the two RRMs. However, only N-terminal RRM (RRM1) was crystallized and its structure was determined at 1.7 Å resolution. It appears that the RRM1 and RRM2 separated before crystallization. To demonstrate which factors affect the separate RRM1-2, we performed limited proteolysis using trypsin. The results indicated that the intact proteins were cleaved by unknown proteases that were associated with them prior to crystallization. In comparison with each of the monomers, the conformations of the β2-β3 loops were highly variable. Furthermore, a comparison with the RRM1-2 structures of HuR and hnRNP A1 revealed that a dimer of RRM1 could be one of the possible conformations of RRM1-2. Our data may provide a guidance for further structural investigations of AUF1 tandem RRM repeat and its mode of ARE binding.
Highlights
Regulation of mRNA stability is one of the most important mechanisms of gene expression control, and it is related to various biological processes including the cell cycle, inflammation, oncogenesis, and apoptosis [1, 2]
We unexpectedly observed the crystallographic dimer of RRM1, comparison with other
Since the two AU-rich element binding/degradation factor 1 (AUF1)-p37 constructs contain two tandem RNA recognition motifs (RRM), we considered the possibility of nonspecific bindings of endogenous E. coli RNAs
Summary
Regulation of mRNA stability is one of the most important mechanisms of gene expression control, and it is related to various biological processes including the cell cycle, inflammation, oncogenesis, and apoptosis [1, 2]. AREs are the most widespread cis-regulatory elements; they have a core AUUUA pentameric sequence within a U-rich region, and their size varies between 40 and 150 nucleotides [4] They function by forming ribonucleoprotein (RNP) complexes with a series of ARE-binding factors. Overexpression of AUF1-p37 selectively degrades ARE-containing mRNA in various cells, while the p40 isoform positively regulates interleukin-10 expression in monocytes [17, 18]. Structural information on AUF1 is currently very limited, each of the N- and Cterminal RRM structures has been determined by NMR spectroscopy [21, 22] Since both RRM1 and RRM2 should be involved in ARE binding, a structure containing both tandem N- and C-terminal RRMs is necessary for the investigation of the mechanism by which AUF1 binds to ARE.
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