Zeyuan Guan

Structural basis of N6-adenosine methylation by the METTL3-METTL14 complex

Chemical modifications of RNA have essential roles in a vast range of cellular processes. N6-methyladenosine (m6A) is an abundant internal modification in messenger RNA and long non-coding RNA that can be dynamically added and removed by RNA methyltransferases (MTases) and demethylases, respectively. An MTase complex comprising methyltransferase-like 3 (METTL3) and methyltransferase-like 14 (METTL14) efficiently catalyses methyl group transfer. In contrast to the well-studied DNA MTase, the exact roles of these two RNA MTases in the complex remain to be elucidated. Here we report the crystal structures of the METTL3–METTL14 heterodimer with MTase domains in the ligand-free, S-adenosyl methionine (AdoMet)-bound and S-adenosyl homocysteine (AdoHcy)-bound states, with resolutions of 1.9, 1.71 and 1.61 Å, respectively. Both METTL3 and METTL14 adopt a class I MTase fold and they interact with each other via an extensive hydrogen bonding network, generating a positively charged groove. Notably, AdoMet was observed in only the METTL3 pocket and not in METTL14. Combined with biochemical analysis, these results suggest that in the m6A MTase complex, METTL3 primarily functions as the catalytic core, while METTL14 serves as an RNA-binding platform, reminiscent of the target recognition domain of DNA N6-adenine MTase. This structural information provides an important framework for the functional investigation of m6A.

Structural basis for specific single-stranded RNA recognition by designer pentatricopeptide repeat proteins

As a large family of RNA-binding proteins, pentatricopeptide repeat (PPR) proteins mediate multiple aspects of RNA metabolism in eukaryotes. Binding to their target single-stranded RNAs (ssRNAs) in a modular and base-specific fashion, PPR proteins can serve as designable modules for gene manipulation. However, the structural basis for nucleotide-specific recognition by designer PPR (dPPR) proteins remains to be elucidated. Here, we report four crystal structures of dPPR proteins in complex with their respective ssRNA targets. The dPPR repeats are assembled into a right-handed superhelical spiral shell that embraces the ssRNA. Interactions between different PPR codes and RNA bases are observed at the atomic level, revealing the molecular basis for the modular and specific recognition patterns of the RNA bases U, C, A and G. These structures not only provide insights into the functional study of PPR proteins but also open a path towards the potential design of synthetic sequence-specific RNA-binding proteins.

MORF9 increases the RNA-binding activity of PLS-type pentatricopeptide repeat protein in plastid RNA editing

RNA editing is a post-transcriptional process that modifies the genetic information on RNA molecules. In flowering plants, RNA editing usually alters cytidine to uridine in plastids and mitochondria. The PLS-type pentatricopeptide repeat (PPR) protein and the multiple organellar RNA editing factor (MORF, also known as RNA editing factor interacting protein (RIP)) are two types of key trans-acting factors involved in this process. However, how they cooperate with one another remains unclear. Here, we have characterized the interactions between a designer PLS-type PPR protein (PLS)3PPR and MORF9, and found that RNA-binding activity of (PLS)3PPR is drastically increased on MORF9 binding. We also determined the crystal structures of (PLS)3PPR, MORF9 and the (PLS)3PPR–MORF9 complex. MORF9 binding induces significant compressed conformational changes of (PLS)3PPR, revealing the molecular mechanisms by which MORF9-bound (PLS)3PPR has increased RNA-binding activity. Similarly, increased RNA-binding activity is observed for the natural PLS-type PPR protein, LPA66, in the presence of MORF9. These findings significantly expand our understanding of MORF function in plant organellar RNA editing.

Corrigendum: Structural basis of N6-adenosine methylation by the METTL3–METTL14 complex

This corrects the article DOI: 10.1038/nature18298

Structural basis of prokaryotic NAD-RNA decapping by NudC

A r g 8 4 Gl u 1 6 2 V a l 1 6 3 Gl y 1 6 4 Gl n 1 6 9 L e u 1 6 7 Gl u 1 6 8 I l e 1 2 7 Gl n 1 2 6 P r o 1 2 5 T y r 1 2 4 T y r 1 2 3 Gl u 1 2 1 T y r 1 0 0

Crystal structure of Cry6Aa: A novel nematicidal ClyA-type α-pore-forming toxin from Bacillus thuringiensis

Crystal (Cry) proteins from Bacillus thuringiensis (Bt) are globally used in agriculture as proteinaceous insecticides. Numerous crystal structures have been determined, and most exhibit conserved three-dimensional architectures. Recently, we have identified a novel nematicidal mechanism by which Cry6Aa triggers cell death through a necrosis-signaling pathway via an interaction with the host protease ASP-1. However, we found little sequence conservation of Cry6Aa in our functional study. Here, we report the 1.90 angstrom (Å) resolution structure of the proteolytic form of Cry6Aa (1-396), determined by X-ray crystallography. The structure of Cry6Aa is highly similar to those of the pathogenic toxin family of ClyA-type α-pore-forming toxins (α-PFTs), which are characterized by a bipartite structure comprising a head domain and a tail domain, thus suggesting that Cry6Aa exhibits a previously undescribed nematicidal mode of action. This structure also provides a framework for the functional study of other nematicidal toxins.

DapF stabilizes the substrate-favoring conformation of RppH to stimulate its RNA-pyrophosphohydrolase activity in Escherichia coli.

Abstract mRNA decay is an important strategy by which bacteria can rapidly adapt to their ever-changing surroundings. The 5′-terminus state of mRNA determines the velocity of decay of many types of RNA. In Escherichia coli, RNA pyrophosphohydrolase (RppH) is responsible for the removal of the 5′-terminal triphosphate from hundreds of mRNAs and triggers its rapid degradation by ribonucleases. A diaminopimelate epimerase, DapF, can directly interact with RppH and stimulate its hydrolysis activity in vivo and in vitro. However, the molecular mechanism remains to be elucidated. Here, we determined the complex structure of DapF–RppH as a heterotetramer in a 2:2 molar ratio. DapF-bound RppH exhibits an RNA-favorable conformation similar to the RNA-bound state, suggesting that association with DapF promotes and stabilizes RppH in a conformation that facilitates substrate RNA binding and thus stimulates the activity of RppH. To our knowledge, this is the first published structure of an RNA–pyrophosphohydrolysis complex in bacteria. Our study provides a framework for further investigation of the potential regulators involved in the RNA–pyrophosphohydrolysis process in prokaryotes.

Structural Insights into the Substrate Recognition Mechanism of Arabidopsis GPP-Bound NUDX1 for Noncanonical Monoterpene Biosynthesis

In plants, monoterpenes are a large family of volatiles that play essential roles in communicating with the surrounding environment, including pollinator attraction, pathogen defense, and plant–plant interactions (Sun et al., 2016). Dozens of monoterpene derivatives have been widely applied in the pharmaceutical, nutraceutical, flavor, and fragrance industries. Because of their high economic value, the biosynthesis of monoterpenes has been thoroughly studied (Degenhardt et al., 2009; Vranova et al., 2013).

Structural basis of the arbitrium peptide–AimR communication system in the phage lysis–lysogeny decision

A bacteriophage can replicate and release virions from a host cell in the lytic cycle or switch to a lysogenic process in which the phage integrates itself into the host genome as a prophage. In Bacillus cells, some types of phages employ the arbitrium communication system, which contains an arbitrium hexapeptide, the cellular receptor AimR and the lysogenic negative regulator AimX. This system controls the decision between the lytic and lysogenic cycles. However, both the mechanism of molecular recognition between the arbitrium peptide and AimR and how downstream gene expression is regulated remain unknown. Here, we report crystal structures for AimR from the SPbeta phage in the apo form and the arbitrium peptide-bound form at 2.20 Å and 1.92 Å, respectively. With or without the peptide, AimR dimerizes through the C-terminal capping helix. AimR assembles a superhelical fold and accommodates the peptide encircled by its tetratricopeptide repeats, which is reminiscent of RRNPP family members from the quorum-sensing system. In the absence of the arbitrium peptide, AimR targets the upstream sequence of the aimX gene; its DNA binding activity is prevented following peptide binding. In summary, our findings provide a structural basis for peptide recognition in the phage lysis–lysogeny decision communication system.Crystal structures of the AimR from SPβ phage in the apo form and the arbitrium peptide-bound form provide a structural basis for peptide recognition in the phage lysis–lysogeny decision communication system.

Structural insights into operator recognition by BioQ in the Mycobacterium smegmatis biotin synthesis pathway.

BACKGROUND Biotin is an essential cofactor in living organisms. The TetR family transcriptional regulator (TFTR) BioQ is the main regulator of biotin synthesis in Mycobacterium smegmatis. BioQ represses the expression of its target genes by binding to a conserved palindromic DNA sequence (the BioQ operator). However, the mechanism by which BioQ recognizes this DNA element has not yet been fully elucidated. METHODS/RESULTS We solved the crystal structures of the BioQ homodimer in its apo-form and in complex with its specific operator at 2.26 Å and 2.69 Å resolution, respectively. BioQ inserts the N-terminal recognition helix of each protomer into the corresponding major grooves of its operator and stabilizes the formation of the complex via electrostatic interactions and hydrogen bonding to induce conformational changes in both the DNA and BioQ. The DNA interface of BioQ is rich in positively charged residues, which help BioQ stabilize DNA binding. We elucidated the structural basis of DNA recognition by BioQ for the first time and identified the amino acid residues responsible for DNA binding via further site-directed mutagenesis. GENERAL SIGNIFICANCE Our findings clearly elucidate the mechanism by which BioQ recognizes its operator in the biotin synthesis pathway and reveal the unique structural characteristics of BioQ that are distinct from other TFTR members.