Over 140 RNA modifications have been discovered, yet only recently have they been studied in depth due to recent technological advancements. N6-methyladenosine (m6A) is an abundant RNA modification in messenger RNA (mRNA) and long non-coding RNA that affects various cellular functions such as mRNA stability, phase-separation of RNAs, and others. Methyltransferase-like protein 16 (METTL16) is one of four catalytically active m6A RNA methyltransferases in humans. Two well-known methylation targets of METTL16 are U6 spliceosomal RNA and hairpins in the 3 untranslated region of MAT2A mRNA. However, METTL16 binds to many other RNAs, including the 3 triple helix of MALAT1. Using in vitro assays, we have started to investigate the kinetic mechanism and other fundamental properties of METTL16. Thus far, we have determined that METTL16 is a monomer in complex with either U6 snRNA or the MALAT1 triple helix. The METTL16•RNA complex has a dissociation constant (KD) of 18 nM with the U6 snRNA and 31 nM with the MALAT1 triple helix. The apparent dissociation constant for S-adenosylmethionine (SAM), the methyl donor, with the METTL16•U6 snRNA binary complex is 112 µM. Under in vitro conditions, the cancer-associated MALAT1 triple helix is not a substrate of METTL16 at position A8290 and other adenosine residues. Preincubation assays suggest that there is an ordered mechanism by which U6 snRNA binds to METTL16 before SAM. Steady-state assays established a kcat of 0.074 min-1 and single-turnover assays established a kchem of 0.56 min-1. This difference in the rates suggests conformational rearrangements and/or product release may be rate limiting. Ongoing work includes the characterization of METTL16 mutants. Mutations in the METTL16 K-loop led to a 7-fold increase of SAM binding to METTL16•U6 snRNA. Future studies will focus on more METTL16 mutants, including critical residues in other structures and those identified in cancer patients, to ascertain how these residues affect the kinetic mechanism of METTL16. This study will enable future research on METTL16 as a therapeutic target.
Chemistry-Biochemistry-Biology Interface (CBBI) Predoctoral Fellowship NIH Grant R00GM111430 amp;amp; R35GM133696 Clare Boothe Luce Program.
