Summary N6-methyladenosine (m6A) is a prevalent, reversible chemical modification of functional RNAs, and is important for central events in biology. The core m6A writers are Mettl3 and Mettl14, which both contain methyltransferase domains. How Mettl3 and Mettl14 cooperate to catalyze methylation of adenosines has remained elusive. We present crystal structures of the complex of Mettl3/Mettl14 methyltransferase domains in apo form as well as with bound S-adenosylmethionine (SAM) or S-adenosylhomocysteine (SAH) in the catalytic site. We determine that the heterodimeric complex of methyltransferase domains, combined with CCCH motifs constitute the minimally required regions for creating m6A modifications in vitro. We also show that Mettl3 is the catalytically active subunit while Mettl14 plays a structural role critical for substrate recognition. Our model provides a molecular explanation for why certain mutations of Mettl3 and Mettl14 lead to impaired function of the methyltransferase complex.

S-adenosylmethionine (SAM) is an essential metabolite that acts as a cofactor for most methylation events in the cell. The N-methyladenosine (mA) methyltransferase METTL16 controls SAM homeostasis by regulating the abundance of SAM synthetase MAT2A mRNA in response to changing intracellular SAM levels. Here we present crystal structures of METTL16 in complex with MAT2A RNA hairpins to uncover critical molecular mechanisms underlying the regulated activity of METTL16. The METTL16-RNA complex structures reveal atomic details of RNA substrates that drive productive methylation by METTL16. In addition, we identify a polypeptide loop in METTL16 near the SAM binding site with an autoregulatory role. We show that mutations that enhance or repress METTL16 activity in vitro correlate with changes in MAT2A mRNA levels in cells. Thus, we demonstrate the structural basis for the specific activity of METTL16 and further suggest the molecular mechanisms by which METTL16 efficiency is tuned to regulate SAM homeostasis.

SUMMARY Late stage 40S ribosome assembly is a highly regulated, dynamic process that occurs in the cytoplasm, alongside the full translation machinery. Seven assembly factors (AFs) regulate and facilitate maturation, but the mechanisms through which they work remain undetermined. Here, we present a series of structures of the immature small subunit (pre-40S) determined by three-dimensional (3D) cryogenic electron microscopy with 3D sorting to assess the molecule’s heterogeneity. These structures demonstrate extensive structural heterogeneity of interface AFs that likely regulates subunit joining during 40S maturation. We also present structural models for the beak and the platform, two regions where the low resolution of previous studies did not allow for localization of AFs, and the rRNA, respectively. These models are supported by biochemical analyses using point variants and suggest that maturation of the 18S 3’-end is regulated by dissociation of the AF Dim1 from the subunit interface, consistent with previous biochemical analyses.

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Chemical modification of RNAs is important for post-transcriptional gene regulation. The METTL3-METTL14 complex generates most N6-methyladenosine (m6A) modifications in mRNAs, and dysregulated methyltransferase expression has been linked to numerous cancers. Here we show that changes in m6A modification location can impact oncogenesis. A gain-of-function missense mutation found in cancer patients, METTL14R298P, promotes malignant cell growth in culture and in transgenic mice. The mutant methyltransferase preferentially modifies noncanonical sites containing a GGAU motif and transforms gene expression without increasing global m6A levels in mRNAs. The altered substrate specificity is intrinsic to METTL3-METTL14, helping us to propose a structural model for how the METTL3-METTL14 complex selects the cognate RNA sequences for modification. Together, our work highlights that sequence-specific m6A deposition is important for proper function of the modification and that noncanonical methylation events can impact aberrant gene expression and oncogenesis.

MicroRNAs (miRNA) are small, non‐coding RNAs that play a critical role in post‐transcriptional gene regulation by acting as a guide for directing the repressor protein Argonaute 2 (Ago2) and its RNA‐induced silencing complex (RISC) to mRNA targets. Recently, we reported on the dynamic movements and localizations of individual miRNAs in living cells, thus demonstrating the feasibility and potential of using single molecule analysis to study non‐coding RNAs in the cellular environment. Our goal is to now extend these studies to the Ago2 protein, the central effecter of the miRNA pathway. Rudell et al (2011) recently found that mutation of the conserved tyrosine Y529 located in the 5′‐end‐binding pocket of Ago2 to a negatively charged glutamate mimics phosphorylation by an as‐yet‐unknown kinase and inhibits small RNA binding. We plan to incorporate this mutation using site‐directed mutagenesis into a plasmid encoding a human Ago2‐EGFP fusion. Using fluorescence microscopy, we will then monitor and compare the localizations and diffusive properties of EGFP‐Ago2 Y529E to its wild‐type counterpart in HeLa cell lines under transient expression. Since EGFP‐Ago2 Y529E is incapable of binding miRNA, it will enable us to distinguish the diffusive motions of miRNA‐loaded RISC from unloaded‐RISC, and to characterize and potentially quantify miRNA loading, a critical step in the miRNA pathway.