ABSTRACT
Chemical modification of RNAs is important for post-transcriptional gene regulation. The METTL3-METTL14 complex generates most N 6 -methyladenosine (m 6 A) modifications in mRNAs, and dysregulated methyltransferase expression has been linked to numerous cancers. Here we show that changes in m 6 A modification location can impact oncogenesis. A gain-of-function missense mutation found in cancer patients, METTL14 R298P , 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 m 6 A 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 m 6 A deposition is important for proper function of the modification and that noncanonical methylation events can impact aberrant gene expression and oncogenesis.
ABSTRACT
In two recent publications in Molecular Cell,Boulias et al. (2019) and Sendinc et al. (2019) use complementary approaches to map m6Am modification sites transcriptome-wide and demonstrate that m6Am can repress translation while increasing the stability of a subset of low-abundance transcripts.
Subject(s)
Protein Processing, Post-Translational , Transcriptome , Methylation , RNA, MessengerABSTRACT
S-adenosylmethionine (SAM) is an essential metabolite that acts as a cofactor for most methylation events in the cell. The N6-methyladenosine (m6A) 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.
Subject(s)
Methyltransferases/metabolism , Methyltransferases/ultrastructure , 3' Untranslated Regions , Adenosine/analogs & derivatives , Binding Sites , HEK293 Cells , Homeostasis , Humans , Methionine Adenosyltransferase/metabolism , Methylation , Methyltransferases/physiology , RNA , RNA, Messenger , RNA, Small Nuclear/metabolism , S-Adenosylmethionine/metabolismABSTRACT
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) cryoelectron microscopy with 3D sorting to assess the molecule's heterogeneity. These structures demonstrate an 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.
Subject(s)
Genetic Heterogeneity , Ribosomal Proteins/chemistry , Ribosome Subunits, Small, Eukaryotic/ultrastructure , Saccharomyces cerevisiae/ultrastructure , Amino Acid Motifs , Binding Sites , Cloning, Molecular , Cryoelectron Microscopy , Crystallography, X-Ray , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Humans , Models, Molecular , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Protein Binding , Protein Interaction Domains and Motifs , Protein Structure, Secondary , RNA, Ribosomal/chemistry , RNA, Ribosomal/genetics , RNA, Ribosomal/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Ribonucleoprotein, U5 Small Nuclear/chemistry , Ribonucleoprotein, U5 Small Nuclear/genetics , Ribonucleoprotein, U5 Small Nuclear/metabolism , Ribosomal Proteins/genetics , Ribosomal Proteins/metabolism , Ribosome Subunits, Small, Eukaryotic/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , ThermodynamicsABSTRACT
N(6)-methyladenosine (m(6)A) is a prevalent, reversible chemical modification of functional RNAs and is important for central events in biology. The core m(6)A 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, constitutes the minimally required regions for creating m(6)A 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.
