NMR Characterization of HuR-RNA Interactions

Proteins for NMR were dissolved in NMR buffer (100 mM NaCl, 10 mM NaPO₄ pH 6.8, 10% D₂O). ¹⁵N NMR data were acquired using Bruker Avance 800 MHz spectrometer with a TCI cryoprobe. ILV ¹³C NMR data were acquired using on a Bruker Avance III 600 MHz spectrometer. NMR data were acquired at 25°C, processed using NMRPipe [29 (link)] and analyzed using NMRView [30 (link)]. For ¹⁵N and ILV chemical shift mapping, 80 μM ¹⁵N/ILV-labeled HuR RRM1/2 was titrated with unlabeled ARE^c-fos RNA at increasing molar ratios of 1:0, 1:0.5, 1:1, and 1:1.7. AZA-9 was titrated at 1:1 and 1:2 molar ratios into 50 μM ¹⁵N/ ILV-labeled HuR RRM1/2 in NMR buffer with 10% d₆-DMSO. Higher protein concentrations or higher molar ratios of AZA-9 resulted in sample precipitation. The ¹⁵N titrations were monitored by acquiring 2D ¹H-¹⁵N TROSY spectra, and the ILV titrations were monitored by acquiring 2D ¹H-¹³C HSQC spectra. The published backbone amide assignments of HuR RRM1/2 (BMRB entry 26628) [20 (link)] and the ILV assignments made herein were used in the NMR analysis.