Structural And Functional Studies Of RNA M~6A Demethylase Alkbh5

N6-methyladenosine (m6A) is the most ubiquitous and abundant modification present in mRNA and long non-coding RNA across eukaryotes, which has been found to function in various pathways of RNA metabolism including mRNA splicing, nuclear export, translation and degradation and to play a vital role in the regulation of gene expression. The m6A modification is involved in diverse life activities such as embryo development, cell apoptosis, spermatogenesis and circadian clock. As a reversible and dynamic modification, m6A methylation is co-regulated by methyltransferases and demethylases. The human AlkB homolog5(hAlkbh5) is the second mRNA m6A demethylase identified after fat mass and obesity associated protein (FTO) recently, playing important regulatory roles in many biological processes including RNA processes and spermatogenesis in mice. Like FTO, Alkbh5is a Fe2+and α-KG (a-ketoglutarate)-dependent non-heme oxygenase, belonging to the AlkB family. Different from the substrate specificity of other AlkB family members, Alkbh5only displays demethylase activity toward the m6A modification in single-stranded nucleic acids. So far, the molecular mechanisms of the specific m6A recognition and single-stranded substrate selection by Alkbh5remain obscure. To elucidate the opened questions, in-depth studies were conducted in this thesis by means of structural biology combined with biochemistry and molecular biology.Through extensive screening of Alkbh5truncated variants and optimization for expression and purification conditions, the human Alkbh566-292protein of high purity and stability was obtained in this study. In vitro demethylation activity assay revealed that the human Alkbh566-292retained the full demethylation activity. Five high resolution crystals of the human Alkbh5catalytic core in complex with citrate and acetate, Mn2+, Mn2+and a-KG, Mn2+and N-oxalylglycine (NOG), Mn2+and pyridine2,4-dicarboxylate (PDCA) were acquired by vapor diffusion in hanging or sitting drops. Determined by molecular replacement, the Alkbh566-292structure consists of seven a-helices, nine β-sheets and several random coils, the active site of which is mainly composed of a highly conserved double-stranded β-helix fold. Structural comparison between Alkbh566-292and other AlkB proteins revealed that the nucleotide recognition lid of Alkbh566-292shows distinctive composition of secondary structures and spatial conformation, which likely confers the substrate selectivity characteristic of Alkbh5. The structure-guided mutagenesis and in vitro demethylase assays proved that the four residues R130, K132, Y139and Y141located at this region are vital for Alkbh5substrate recognition and binding, which may selectively bind m6A through hydrogen bonds, electrostatic and π-π stacking interactions. The structural alignment also shows that Alkbh5harbors a unique disulfide bond between C230and C267. The disulfide bond in Alkbh5is highly conserved in many different species but is not shared by other AlkB family members. When the double-stranded DNA from other AlkB family complex structures was modeled onto the catalytic site of Alkbh5, the Flip3motif of Alkbh5sterically clashed with the unmethylated strand, impeding the access of dsDNA and dsRNA to the active site of Alkbh5. Both the electrophoretic mobility shift and in vitro demethylase assays supported the stronger dsDNA binding affinity and the higher dsDNA demethylation activity in the C230S mutant, indicating that this disulfide bond is the structural element that determines the single-stranded preference of Alkbh5. Our work also investigated the inhibition of Alkbh5demethylase activity by four a-KG analogs including NOG, PDCA, succinate and citrate. We found that, in contrast to FTO, Alkbh5has potent binding preference towards smaller molecule inhibitors that is likely caused by the smaller active site cavity of Alkbh5. Finally, based on the electrostatic potential map of Alkbh5, we proposed an ssRNA binding model of Alkbh5according to the results of site-directed mutagenesis of the surface residues together with demethylation assays.Taken together, our studies provide a structural basis for understanding the selective substrate recognition and binding of Alkbh5and offer a foundation for selective drug design against AlkB members.