RNA recognition by the ADAR2 dsRBMs: a 50 kDa protein-RNA complex by NMR

Richard Stefl¹, Lenka Skrisovska¹, Ming Xu², Ronald B. Emeson², Frederic Allain¹

¹Institute of Molecular Biology and Biophysics, ETH Zurich, 8093 Zurich

²Department of Pharmacology, Vanderbilt University, Nashville, Tennessee 37232, USA

Adenosine deaminases that act on RNA (ADARs) tune and regulate gene expression. Although ADARs act mostly as nonspecific enzymes, they can recode certain genes in a highly specific manner. This results from preferential binding of the ADARs to certain RNA substrates. To understand how ADARs bind RNA, we investigate the N-terminal region of ADAR2 in complex with a 71 nucleotide RNA encoding the R/G site of the GluR-B (MW ~50 kDa), using nuclear magnetic resonance (NMR) spectroscopy. We chose solution-state NMR technique as a number of groups failed in making crystals of ADAR-RNA complexes. The studied complex represents a challenge for structure determination by NMR because of its size and elongated shape. However, we identified that two structured domains (double-stranded RNA-binding domains, dsRBMs) located in N-terminal region of ADAR2 bind RNA substrate in independent manner, each domain binding a different site on RNA. This allowed us to make two subcomplexes in which all necessary NMR experiments for structural determination could be measured. The full-length complex is being reconstructed using long-range structural information derived from residual dipolar couplings (RDCs) measured on a 50 kDa ADAR-RNA complex. This study demonstrates how NMR can be used for structural determination of large protein-RNA complexes, provided that rationale design of studied constructs along with deuteration and TROSY techniques are used.