Abstract
AU-rich element RNA-binding protein 1 (AUF1) regulates the stability and/or translational efficiency of diverse mRNA targets, including many encoding products controlling the cell cycle, apoptosis, and inflammation by associating with AU-rich elements residing in their 3'-untranslated regions. Previous biochemical studies showed that optimal AUF1 binding requires 33-34 nucleotides with a strong preference for U-rich RNA despite observations that few AUF1-associated cellular mRNAs contain such extended U-rich domains. Using the smallest AUF1 isoform (p37(AUF1)) as a model, we employed fluorescence anisotropy-based approaches to define thermodynamic parameters describing AUF1 ribonucleoprotein (RNP) complex formation across a panel of RNA substrates. These data demonstrated that 15 nucleotides of AU-rich sequence were sufficient to nucleate high affinity p37(AUF1) RNP complexes within a larger RNA context. In particular, p37(AUF1) binding to short AU-rich RNA targets was significantly stabilized by interactions with a 3'-purine residue and largely base-independent but non-ionic contacts 5' of the AU-rich site. RNP stabilization by the upstream RNA domain was associated with an enhanced negative change in heat capacity consistent with conformational changes in protein and/or RNA components, and fluorescence resonance energy transfer-based assays demonstrated that these contacts were required for p37(AUF1) to remodel local RNA structure. Finally, reporter mRNAs containing minimal high affinity p37(AUF1) target sequences associated with AUF1 and were destabilized in a p37(AUF1)-dependent manner in cells. These findings provide a mechanistic explanation for the diverse population of AUF1 target mRNAs but also suggest how AUF1 binding could regulate protein and/or microRNA binding events at adjacent sites.
Highlights
Using the smallest AUF1 isoform (p37AUF1) as a model, we employed fluorescence anisotropy-based approaches to define thermodynamic parameters describing AUF1 ribonucleoprotein (RNP) complex formation across a panel of RNA substrates. These data demonstrated that 15 nucleotides of AU-rich sequence were sufficient to nucleate high affinity p37AUF1 RNP complexes within a larger RNA context
P37AUF1 Binding to Short (Յ 16-nt) AU-rich elements (AREs) Substrates Is Dramatically Stabilized by the Addition of Flanking non-ARE Sequences—Previously we showed that p37AUF1 dimers bind sequentially with high affinity to ARE-based RNA substrates of 34 nt or more in length [12]
The nucleotide determinants required for AUF1 binding to RNA targets control the affinity and positioning of AUF1 recruitment and by extension the biochemical and functional consequences of these interactions
Summary
Consistent with this, screening efforts have identified hundreds of AUF1-targeted mRNAs among the cellular transcript population but with few containing 3Ј-UTR sequences of 30 nt that consist solely of A and U residues [25, 26] and to date have not delineated precise RNA requirements for high affinity AUF1 binding Together these data suggest that only a subset of AUF1-associated RNA nucleotides need be AU-rich to nucleate assembly of functional RNP complexes. Fluorescence-based assay systems permitted quantitative assessment of the thermodynamic contributions of RNA ligand subdomains to AUF1 RNP assembly and local RNA structural remodeling, whereas cultured cell models were used to determine whether minimal high affinity AUF1 binding sites were functional Findings from these experiments provide an explanation for the 3Ј-UTR sequence heterogeneity observed among AUF1-targeted mRNAs and suggest potential mechanisms for cooperative or competitive relationships between AUF1 and other trans-acting factors for mRNA targets
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
