Tingting Zou

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.

Mycobacterium smegmatis BioQ defines a new regulatory network for biotin metabolism.

Biotin (vitamin H), the sulfur‐containing enzyme cofactor, is an essential micronutrient for three domains of life. Given the fact that biotin is an energetically expensive molecule whose de novo biosynthesis demands 20 ATP equivalents each, it is reasonable that bacteria have evolved diversified mechanisms in various microorganisms to tightly control biotin metabolism. Unlike the Escherichia coli BirA, the prototypical bi‐functional version of biotin protein ligase (BPL) in that it acts as a repressor for biotin biosynthesis pathway, the BirA protein of Mycobacterium smegmatis (M. smegmatis), a closely relative of the tuberculosis‐causing pathogen, Mycobacterium tuberculosis, lacked the DNA‐binding activity. It raised a possibility that an alternative new regulator might be present to compensate the loss of regulatory function. Here we report that this is the case. Genomic context analyses of M. smegmatis detected a newly identified BioQ homolog classified into the TetR family of transcription factor and its recognizable palindromes. The M. smegmatis BioQ protein was overexpressed and purified to homogeneity. Size‐exclusion chromatography combined with chemical cross‐linking studies demonstrated that the BioQ protein had a propensity to dimerize. The promoters of bioFD and bioQ/B were mapped using 5′‐RACE. Electrophoretic mobility shift assays revealed that BioQ binds specifically to the promoter regions of bioFD and bioQ/B. Further DNase I foot‐printing elucidated the BioQ‐binding palindromes. Site‐directed mutagenesis suggested the important residues critical for BioQ/DNA binding. The isogenic mutant of bioQ (ΔbioQ) was generated using the approach of homologous recombination. The in vivo data from the real‐time qPCR combined with the lacZ transcriptional fusion experiments proved that removal of bioQ gave significant increment with expression of bio operons. Also, expression of bio operons were repressed by exogenous addition of biotin, and this repression seemed to depend on the presence of BioQ protein. Thereby, we believed that M. smegmatis BioQ is not only a negative auto‐regulator but also a repressor for bioFD and bioB operons involved in the biotin biosynthesis pathway. Collectively, this finding defined the two‐protein paradigm of BirA and BioQ, representing a new mechanism for bacterial biotin metabolism.

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.

Specific RNA Recognition by Designer Pentatricopeptide Repeat Protein

Manipulation of gene expression through targeting specific DNA or RNA sequences is a significant challenge. In the past decade, transcription activator-like (TAL) effectors and zinc fingers (ZFs) have been successfully developed into useful tools for DNA recognition (Bogdanove and Voytas, 2011; Deng et al., 2012a, 2012b). However, little progress has been made in the realm of RNA targeting due to the lack of understanding about the modular RNA recognition mechanism. Pumilio and FBF homology (PUF) proteins and pentatricopeptide repeat (PPR) proteins are two types of sequence-specific single-strand RNA (ssRNA) binding proteins with the potential to serve as effective RNA targeting tools (Filipovska and Rackham, 2013; Campbell et al., 2014).

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

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Human m6A writers: Two subunits, 2 roles

ABSTRACT Cellular RNAs with diverse chemical modifications have been observed, and N6-methyladenosine (m6A) is one of the most abundant internal modifications found on mRNA and non-coding RNAs, playing a vital role in diverse biologic processes. In humans, m6A modification is catalyzed by the METTL3-METTL14 methyltransferase complex, which is regulated by WTAP and another factor. Three groups have recently and independently reported the structure of this complex with or without cofactors. Here, we focus on the detailed mechanism of the m6A methyltransferase complex and the properties of each subunit. METTL3 is predominantly catalytic, with a function reminiscent of N6-adenine DNA methyltransferase systems, whereas METTL14 appears to be a pseudomethyltransferase that stabilizes METTL3 and contributes to target RNA recognition. The structural and biochemical characterization of the METTL3-METTL14 complex is a major step toward understanding the function of m6A modification and developing m6A-related therapies.

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.

Solution structure of the RNA recognition domain of METTL3-METTL14 N6-methyladenosine methyltransferase

ABSTRACTN6-methyladenosine (m6A), a ubiquitous RNA modification, is installed by METTL3-METTL14 complex. The structure of the heterodimeric complex between the methyltransferase domains (MTDs) of METTL3 and METTL14 has been previously determined. However, the MTDs alone possess no enzymatic activity. Here we present the solution structure for the zinc finger domain (ZFD) of METTL3, the inclusion of which fulfills the methyltransferase activity of METTL3-METTL14. We show that the ZFD specifically binds to an RNA containing 5′-GGACU-3′ consensus sequence, but does not to one without. The ZFD thus serves as the target recognition domain, a structural feature previously shown for DNA methyltransferases, and cooperates with the MTDs of METTL3-METTL14 for catalysis. However, the interaction between the ZFD and the specific RNA is extremely weak, with the binding affinity at several hundred micromolar under physiological conditions. The ZFD contains two CCCH-type zinc fingers connected by an anti-parallel β-sheet. Mutational analysis and NMR titrations have mapped the functional interface to a contiguous surface. As a division of labor, the RNA-binding interface comprises basic residues from zinc finger 1 and hydrophobic residues from β-sheet and zinc finger 2. Further we show that the linker between the ZFD and MTD of METTL3 is flexible but partially folded, which may permit the cooperation between the two domains during catalysis. Together, the structural characterization of METTL3 ZFD paves the way to elucidate the atomic details of the entire process of RNA m6A modification.