Elucidating the Kinetic Mechanism of Human METTL16 and Its Role in Miller-Dieker Syndrome

Over 140 biologically relevant RNA modifications have been discovered to date. The N6-methyladenosine (m6A) RNA modification has had many important roles determined but few methyltransferases have been identified that introduce this modification in humans. This dissertation focuses on one of these methyltransferases, human methyltransferase-like protein 16 (METTL16). Herein, three questions were explored pertaining to METTL16: (i) what is the kinetic mechanism of METTL16, (ii) do the amino acids, domains, and regions in the solved METTL16 structures reflect their suggested functions and (iii) in a disease such as Miller-Dieker Syndrome, where there is half as much METTL16, is the function of METTL16 as a S-adenosylmethionine (SAM) biosensor disrupted and thus, global RNA methyl modifications decreased? METTL16 is a recently discovered SAM-dependent, m6A methyltransferase that methylates U6 small nuclear (snRNA) and binds to the metastasis-associated lung adenocarcinoma transcript 1 (MALAT1). I discovered that METTL16 does not methylate the MALAT1 triple helix RNA in vitro, but METTL16 does have a similar binding affinity for the triple helix compared to the U6 snRNA. I establish a basic kinetic scheme with parameters for METTL16 methylating U6 snRNA that includes the binding order of substrates, dissociation constants and rate constants. Next, I performed structure-guided mutational analysis of METTL16 to understand the functional roles of specific amino acids, regions, and domains within METTL16. For each METTL16 mutant or construct, I obtained rate constants under single-turnover conditions and dissociation constants for the substrates SAM and U6 snRNA; these data confirm structure-function relationships hypothesized by solved METTL16 structures. Several METTL16 cancer mutations were also tested to understand how METTL16 functions might be perturbed in cancer. Lastly, I examined the possible connections between METTL16 and the rare genetic disease Miller-Dieker Syndrome (MDS). MDS results in haploinsufficiency in twenty-six genes including METTL16 due to a ~1.4 megabase pair deletion on a single copy of chromosome 17. I hypothesized that METTL16 mRNA and protein levels would decrease by 2-fold; thus, the function of METTL16 as a SAM biosensor might be disrupted causing alterations in RNA modifications in MDS. METTL16 mRNA and protein levels were shown to be 2-fold less. However, the UPLC-MS/MS experiments indicated no alterations in RNA modification abundances in MDS cells. Overall this dissertation work improves our understanding of the methylation functions of METTL16 by providing many kinetic parameters of WT METTL16 and various variants that elucidate structural components of METTL16. I also started to reveal connections between METTL16 and MDS through various omics approaches that should help find treatments for the disease.