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Gene regulation is tightly controlled at many points, especially at the RNA level. Cis-acting sequences within 3'-untranslated regions (UTRs) often regulate mRNA decay kinetics and translational efficiency. A well-studied cis-acting element is a family of RNA sequences known as AU-rich elements (AREs), which often contain repeated AUUUA motifs and are enriched in the 3' UTRs of mRNAs encoding inflammatory and pro-tumorigenic factors. Many RNA-binding proteins (RBPs) target AREs to regulate mRNA stability and/or translation. One prototypical example is AUF1. AUF1 contains two tandem RNA recognition motifs (RRMs), common RNA-binding domains that stabilize formation of many ribonucleoprotein (RNP) complexes. However, previous data indicated that AUF1's tandem RRM domains only weakly bind RNA, which suggests that flanking protein sequences contribute to RNA binding in some way. Using equilibrium-based, quantitative fluorescence anisotropy-based assays, we validated the weak binding of the tandem RRM domain to ARE substrates. Chemical denaturation experiments eliminated the possibility that flanking protein sequences contribute to the stability of RRM domain folding, prompting the hypothesis that they play direct roles in stabilization of RNP complexes. Using different protein mutants, we then measured the impact of individual flanking domains on RNP assembly with various ARE substrates. We found that the N- and C-terminal domains make distinct contributions to RNP formation, with the C-terminal domain being the primary contributor to the initial RNA:protein binding event, while both flanking domains enhance protein oligomerization on AREs. Focusing primarily on the C-terminal domain, our emerging data suggest that the domain is intrinsically disordered in solution but may undergo a conformational change upon interaction with RNA. This study is supported by the NCI grant R21 CA247647 awarded to Dr. Gerald Wilson and the Nathan Schnaper Intern Program through NCI grant R25 CA186872.
Lee et al. (Fri,) studied this question.