1H, 13C, 15N backbone chemical shift assignments of the extended ARID domain in human AT-rich interactive domain protein 5a (Arid5a)

Abstract

The family of AT-rich interactive domain (ARID) containing proteins -Arids- contains 15 members that have almost exclusively been described as DNA-binding proteins. Interestingly, a decade ago the family member Arid5a was found to bind and stabilize mRNAs of immune system key players and thereby account for driving inflammatory and autoimmune diseases. How exactly binding to DNA and RNA is coordinated by the Arid5a ARID domain remains unknown, mainly due to the lack of atom-resolved information on nucleic acid-binding. This in particular applies to the protein’s ARID domain, despite the comfortable size of its core unit for NMR-based investigations. Furthermore, the core domain of ARID domains is found to be extended by functionally relevant, often flexible stretches, but whether such elongations are present and crucial for the versatile Arid5a functions is unknown. We here provide a near-complete NMR backbone resonance assignment of the Arid5a ARID domain with N- and C-terminal extensions, which serves as a basis for further studies of its nucleic acid-binding preferences and targeted inhibition by means of NMR. Our data thus significantly contribute to unravelling mechanisms of Arid5a-mediated gene regulation and diseases.

Arid5a uses disordered extensions of its core ARID domain for distinct DNA- and RNA-recognition and gene regulation

“…Human Arid5a ARID constructs used in this study were designed and cloned as described previously 31 . In brief, we used different domain boundaries, comprising the minimal ARID core (ARID 49-152 ) alone or extended either N- (ARID 37-152 ) or C- (ARID 49-183 ) terminally, or both (ARID 37-183 ), with the numbers representing the natural sequence in the full-length context ( Fig.…”

“…Proteins were expressed and purified as described recently 31 , with an additional cation-exchange chromatography (CEX) step. An ENrich TM S 5x50 column (Bio-Rad) was equilibrated with low-salt (LS) buffer (20 mM Bis-Tris, 40 mM NaCl, 2 mM DTT, 0.02% NaN 3 , pH 7.2) and subsequently loaded with size-exclusion (SEC) purified protein, buffer-exchanged to LS-buffer in Amicon ® ultra centrifugal filters (MWCO: 3 kDa)and concentrated to 5 ml.…”

“…b) Comparison of human and mouse Arid5a ARID sequences with the human Arid5b ARID, as obtained by Clustal Omega 30 . The ARID secondary structure elements (helices H1 to H6) are indicated above the sequence for human Arid5b (blue, PDB 1IG6) and human Arid5a (red 31 ). For full sequence alignment of Arid5a human with mouse see Suppl.…”