Alternatively expressed domains of AU-rich element RNA-binding protein 1 (AUF1) regulate RNA-binding affinity, RNA-induced protein oligomerization, and the local conformation of bound RNA ligands

AU-rich element RNA-binding protein 1 (AUF1) binding to AU-rich elements (AREs) in the 3'-untranslated regions of mRNAs encoding many cytokines and other regulatory proteins modulates mRNA stability, thereby influencing protein expression. AUF1-mRNA association is a dynamic paradigm directed by various cellular signals, but many features of its function remain poorly described. There are four isoforms of AUF1 that result from alternative splicing of exons 2 and 7 from a common pre-mRNA. Preliminary evidence suggests that the different isoforms have varied functional characteristics, but no detailed quantitative analysis of the properties of each isoform has been reported despite their differential expression and regulation. Using purified recombinant forms of each AUF1 protein variant, we used chemical cross-linking and gel filtration chromatography to show that each exists as a dimer in solution. We then defined the association mechanisms of each AUF1 isoform for ARE-containing RNA substrates and quantified relevant binding affinities using electrophoretic mobility shift and fluorescence anisotropy assays. Although all AUF1 isoforms generated oligomeric complexes on ARE substrates by sequential dimer association, sequences encoded by exon 2 inhibited RNA-binding affinity. By contrast, the exon 7-encoded domain enhanced RNA-dependent protein oligomerization, even permitting cooperative RNA-binding activity in some contexts. Finally, fluorescence resonance energy transfer-based assays showed that the different AUF1 isoforms remodel bound RNA substrates into divergent structures as a function of protein:RNA stoichiometry. Together, these data describe isoform-specific characteristics among AUF1 ribonucleoprotein complexes, which likely constitute a mechanistic basis for differential functions and regulation among members of this protein family.