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oai:keep.lib.asu.edu:node-1981582024-12-23T18:01:48Zoai_pmh:repo_items

198158 https://hdl.handle.net/2286/R.2.N.198158 http://rightsstatements.org/vocab/InC/1.0/ All Rights Reserved 2024 2026-12-01T11:19:27 237 pages Doctoral Dissertation Academic theses Text eng Perry, Kayla Nicole Lapinaite, Audrone Chiu, Po-Lin Mangone, Marco Arizona State University Partial requirement for: Ph.D., Arizona State University, 2024 Field of study: Biochemistry RNA is a dynamic and multifunctional biological compound that temporarily stores genetic information, regulates gene expression, and even catalyzes chemical reactions. These diverse RNA functions are partly supported by chemical modifications: post-transcriptional alterations made to RNA molecules without changing the nucleic acid sequence. The most abundant mRNA modification in eukaryotes, N6- methyladenosine (m6A), is involved in numerous biological processes such as embryonic development, learning, memory formation, circadian rhythms, and immunoregulation. The m6A modification is deposited by a large multiprotein complex comprised of core and accessory subunits. The core complex comprises a heterodimer of Methyltransferase Like 3 (METTL3) and Methyltransferase Like 14 (METTL14) proteins. Together, METTL3-METTL14 catalyzes the m6A methylation reaction at the central adenosine residue in the highly conserved DRACH (D = A/G/U, R = A/G, and H = A/C/U) methylation motif in mRNA. Despite its wide abundance, most motifs remain unmodified. How specific mRNA transcripts and motifs are selected for m6A deposition remains elusive. As a result, the design of effective therapeutic strategies to correct deregulated m6A modifications in pathological states remains limited. Therefore, this study employs biochemical and structural approaches to uncover the molecular mechanism of m6A deposition and RNA recognition by the core complex. A novel in vitro single-turnover methylation kinetic assay reveals the enzyme kinetics of the core complex’s DRACA motif preferences. Additionally, for the first time, the core complex is shown to recognize the RNA sequences flanking the DRACH methylation motif at both 5’ and 3’ ends. Thus, the sequence and length of these flanking ends, with potential crosstalk of the DRACH motif, intrinsically regulate m6A deposition. Truncation constructs of the core complex reveal the domains required for efficient single-stranded RNA (ssRNA) methylation and provide insight into the domains responsible for recognizing RNA’s flanking sequences. Collectively, these findings may suggest a new RNA-binding architecture in the core complex. Overall, this study illuminates the molecular mechanism of m6A deposition and RNA recognition by the core complex and may fuel the rational design of novel therapeutic strategies to treat deregulated m6A modifications in pathological states. Biochemistry Kinetics m6A Methyltransferases Protein RNA Molecular Mechanism of RNA Methylation by Heterodimeric N6-Methyladenosine (m6A) Methyltransferases METTL3 and METTL14