Unstructured protein domains stabilize RNA binding and mediate RNA folding by AUF1.

AUF1 is an RNA-binding protein that targets AU-rich elements (AREs), cis-acting regulatory sequences commonly enriched in mRNAs encoding inflammatory mediators and oncoproteins. AUF1 post-transcriptionally regulates gene expression by modulating the stability and/or translational efficiency of mRNA targets in a context-specific manner; however, the mechanisms by which AUF1 directly engages RNA substrates and mediates regulatory outcomes remain largely unknown. The purpose of this study was to define the biochemical basis for RNA recognition by AUF1 using the smallest protein isoform (p37^AUF1) as a model. AUF1 contains two tandem RNA recognition motifs (RRMs), common RNA-binding domains that stabilize formation of many ribonucleoprotein (RNP) complexes. Using quantitative fluorescence anisotropy-based assays, we observed that p37^AUF1's tandem RRM domain only weakly binds ARE substrates. Testing a panel of protein mutants revealed that the N- and C-terminal flanking domains each make modest but similar contributions to stabilization of both the initial RNA:protein complex and a subsequent protein binding event. However, focused protein truncations showed that residues immediately N-terminal of the RRMs were vital for high affinity binding, but only in the context of the C-terminal domain. The C-terminal domain was also required for protein-induced RNA remodeling; both this function and its RNP-stabilizing role involve non-base-specific contacts with RNA upstream of the AU-rich motif. Finally, our data suggest that the C-terminal domain is intrinsically disordered but may undergo a conformational change upon interaction with RNA ligands. Together, these findings reveal distinct roles for flanking protein domains in RNA binding and remodeling by AUF1.