Protein and RNA Determinants Regulating Binding of AUF1, A Major Post-Transcriptional Regulator of Gene Expression

Abstract

AU-rich element RNA binding protein 1 (AUF1) regulates the stability of mRNAs containing AU-rich elements (AREs) in their 3'-untranslated regions. AREs are highly enriched in mRNAs that encode cytokines, cell cycle regulators, and other regulatory proteins, making AUF1 a critical factor in control of many cellular systems. Four AUF1 isoforms arise through alternative splicing of exons 2 and 7 from a common pre-mRNA. Preliminary evidence suggested that the different isoforms have varied functional and regulatory characteristics, but no detailed, quantitative analysis of their biochemical properties had been reported. Using purified recombinant forms of each AUF1 protein variant, we used chemical crosslinking and gel filtration chromatography to show that each exists as a dimer in solution. We then defined the association mechanisms of each isoform for ARE-containing RNA substrates and quantified binding affinities using electrophoretic mobility shift and fluorescence anisotropy assays. While all AUF1 isoforms generated oligomeric complexes on ARE substrates by sequential dimer association, exon 2-encoded sequences inhibited RNA-binding affinity. By contrast, the exon 7-encoded domain enhanced RNA-dependent protein oligomerization. Finally, fluorescence resonance energy transfer-based assays (FRET) showed that the different 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 (RNP) complexes, which likely constitute a mechanistic basis for differential function and regulation. Subsequently we evaluated RNA requirements for AUF1 binding. Despite a binding site size of 33-34 nucleotides on ARE substrates, p37<super>AUF1</super> requires only 15 nucleotides of AU-rich sequence to form stable RNPs in a larger RNA context. In particular, a 3'-guanine and 5'-base-specific sequence stabilize p37<super>AUF1</super> binding, but by a mechanism that does not involve new ionic contacts. However, van't Hoff and FRET analyses showed that AUF1 contacts with the 5&rsquo-extension contribute to RNA structural condensation. Reporter mRNAs containing minimal high-affinity target sequences associate with AUF1 by RNP-immunoprecipitation and are destabilized in an AUF1-dependent manner in cells. These findings provide a mechanistic explanation for the diverse population of AUF1 target mRNAs, but also suggest that the role of AUF1 and proteins/miRNAs binding adjacent sites may be reciprocally regulated by local RNA structure.