Poster abstracts
Poster number 137 submitted by Sukanya Srinivasan
Auxiliary domains of the mammalian DEAH/RHA helicase DHX36 work in concert with the helicase core domains to remodel RNA structures.
Sukanya Srinivasan (Center for RNA Science and Therapeutics), Zhonghua Liu (Department of Pathology), Tsan Sam Xiao (Department of pathology), Eckhard Jankowsky (Center for RNA Science and Therapeutics)
Abstract:
The DEAH/RHA helicase DHX36 is linked to DNA and RNA G-quadruplex structures and AU-rich element (ARE) - mediated mRNA deadenylation and decay. In vitro, DHX36 resolves DNA and RNA G-quadruplex structures and unwinds duplexes. However, the molecular basis for RNA structure remodeling and important biochemical features of DHX36 substrate specificity remain unclear. To address these questions, we combined structural and biochemical analyses of mouse DHX36. We have solved the crystal structure of DHX36 in complex with the non-hydrolysable ATP analog AMP-PNP and in complex with ADP. The overall structures of mDHX36 closely resemble structures for previously reported bovine and fly DHX36. However, we observe differences to the previous structures, consistent with conformational changes that accompany the different stages of the ATP binding and hydrolysis and the RNA remodeling cycle.
Biochemical experiments demonstrate that at saturating DHX36 concentration, unwinding rate constants are four-fold higher for quadruplexes than for duplex structures. Besides, the functional affinity of DHX36 for quadruplexes is more than 20 fold higher than that for duplex structures. The data thus reveal that DHX36 preferentially binds and remodels RNA quadruplex structures, compared to duplex structures. We further show that the N-terminal DSM domain functions not only as a quadruplex binding adaptor but also promotes the remodeling of both, duplex and quadruplex structures. Besides, we demonstrate that two previously uncharacterized DHX36 regions, a 5’-β-hairpin in the helicase core, and the OB-fold domain are essential for the ability of DHX36 to bind and remodel both, quadruplex and duplex structures. We also show that DHX36 utilizes all nucleotides, but not indiscriminately. Remarkably, ATP at physiological levels negatively regulates DHX36 unwinding of duplexes and positively regulates DHX36 remodeling of quadruplexes. This indicates that the nucleotide selectivity of DHX36 influences its RNA substrate selectivity and vice versa. Collectively, our data reveal that the auxiliary domains of DHX36 work in conjunction with the helicase core to remodel RNA structures.
Keywords: Helicase, G-quadruplexes, RNA