Assembly of Functional Ribonucleoprotein Complexes by AU-rich Element RNA-binding Protein 1 (AUF1) Requires Base-dependent and -independent RNA Contacts

作者：Zucconi Beth E; Wilson Gerald M^*

来源：Journal of Biological Chemistry, 2013, 288(39): 28034-28048.

DOI：10.1074/jbc.M113.489559

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.

• 出版日期2013-9-27