Substrate specificity of Mycobacterium tuberculosis tRNA terminal nucleotidyltransferase toxin MenT3.

Mycobacterium tuberculosis transfer RNA (tRNA) terminal nucleotidyltransferase toxin, MenT3, incorporates nucleotides at the 3'-CCA end of tRNAs, blocking their aminoacylation and inhibiting protein synthesis. Here, we show that MenT3 most effectively adds CMPs to the 3'-CCA end of tRNA. The crystal structure of MenT3 in complex with CTP reveals a CTP-specific nucleotide-binding pocket. The 4-NH2 and the N3 and O2 atoms of cytosine in CTP form hydrogen bonds with the main-chain carbonyl oxygen of P120 and the side chain of R238, respectively. MenT3 expression in Escherichia coli selectively reduces the levels of seryl-tRNASers, indicating specific inactivation of tRNASers by MenT3. Consistently, MenT3 incorporates CMPs into tRNASer most efficiently, among the tested E. coli tRNA species. The longer variable loop unique to class II tRNASers is crucial for efficient CMP incorporation into tRNASer by MenT3. Replacing the variable loop of E. coli tRNAAla with the longer variable loop of M. tuberculosis tRNASer enables MenT3 to incorporate CMPs into the chimeric tRNAAla. The N-terminal positively charged region of MenT3 is required for CMP incorporation into tRNASer. A docking model of tRNA onto MenT3 suggests that an interaction between the N-terminal region and the longer variable loop of tRNASer facilitates tRNA substrate selection.

Structure of the Caenorhabditis elegans m6A methyltransferase METT10 that regulates SAM homeostasis.

In Caenorhabditis elegans, the N6-methyladenosine (m6A) modification by METT10, at the 3'-splice sites in S-adenosyl-l-methionine (SAM) synthetase (sams) precursor mRNA (pre-mRNA), inhibits sams pre-mRNA splicing, promotes alternative splicing coupled with nonsense-mediated decay of the pre-mRNAs, and thereby maintains the cellular SAM level. Here, we present structural and functional analyses of C. elegans METT10. The structure of the N-terminal methyltransferase domain of METT10 is homologous to that of human METTL16, which installs the m6A modification in the 3'-UTR hairpins of methionine adenosyltransferase (MAT2A) pre-mRNA and regulates the MAT2A pre-mRNA splicing/stability and SAM homeostasis. Our biochemical analysis suggested that C. elegans METT10 recognizes the specific structural features of RNA surrounding the 3'-splice sites of sams pre-mRNAs, and shares a similar substrate RNA recognition mechanism with human METTL16. C. elegans METT10 also possesses a previously unrecognized functional C-terminal RNA-binding domain, kinase associated 1 (KA-1), which corresponds to the vertebrate-conserved region (VCR) of human METTL16. As in human METTL16, the KA-1 domain of C. elegans METT10 facilitates the m6A modification of the 3'-splice sites of sams pre-mRNAs. These results suggest the well-conserved mechanisms for the m6A modification of substrate RNAs between Homo sapiens and C. elegans, despite their different regulation mechanisms for SAM homeostasis.

The tRNA pseudouridine synthase TruB1 regulates the maturation of let-7 miRNA.

Let-7 is an evolutionary conserved microRNA that mediates post-transcriptional gene silencing to regulate a wide range of biological processes, including development, differentiation, and tumor suppression. Let-7 biogenesis is tightly regulated by several RNA-binding proteins, including Lin28A/B, which represses let-7 maturation. To identify new regulators of let-7, we devised a cell-based functional screen of RNA-binding proteins using a let-7 sensor luciferase reporter and identified the tRNA pseudouridine synthase, TruB1. TruB1 enhanced maturation specifically of let-7 family members. Rather than inducing pseudouridylation of the miRNAs, high-throughput sequencing crosslinking immunoprecipitation (HITS-CLIP) and biochemical analyses revealed direct binding between endogenous TruB1 and the stem-loop structure of pri-let-7, which also binds Lin28A/B. TruB1 selectively enhanced the interaction between pri-let-7 and the microprocessor DGCR8, which mediates miRNA maturation. Finally, TruB1 suppressed cell proliferation, which was mediated in part by let-7. Altogether, we reveal an unexpected function for TruB1 in promoting let-7 maturation.

Mechanistic insights into tRNA cleavage by a contact-dependent growth inhibitor protein and translation factors.

Contact-dependent growth inhibition is a mechanism of interbacterial competition mediated by delivery of the C-terminal toxin domain of CdiA protein (CdiA-CT) into neighboring bacteria. The CdiA-CT of enterohemorrhagic Escherichia coli EC869 (CdiA-CTEC869) cleaves the 3'-acceptor regions of specific tRNAs in a reaction that requires the translation factors Tu/Ts and GTP. Here, we show that CdiA-CTEC869 has an intrinsic ability to recognize a specific sequence in substrate tRNAs, and Tu:Ts complex promotes tRNA cleavage by CdiA-CTEC869. Uncharged and aminoacylated tRNAs (aa-tRNAs) were cleaved by CdiA-CTEC869 to the same extent in the presence of Tu/Ts, and the CdiA-CTEC869:Tu:Ts:tRNA(aa-tRNA) complex formed in the presence of GTP. CdiA-CTEC869 interacts with domain II of Tu, thereby preventing the 3'-moiety of tRNA to bind to Tu as in canonical Tu:GTP:aa-tRNA complexes. Superimposition of the Tu:GTP:aa-tRNA structure onto the CdiA-CTEC869:Tu structure suggests that the 3'-portion of tRNA relocates into the CdiA-CTEC869 active site, located on the opposite side to the CdiA-CTEC869 :Tu interface, for tRNA cleavage. Thus, CdiA-CTEC869 is recruited to Tu:GTP:Ts, and CdiA-CT:Tu:GTP:Ts recognizes substrate tRNAs and cleaves them. Tu:GTP:Ts serves as a reaction scaffold that increases the affinity of CdiA-CTEC869 for substrate tRNAs and induces a structural change of tRNAs for efficient cleavage by CdiA-CTEC869.

Substrate specificities of Escherichia coli ItaT that acetylates aminoacyl-tRNAs.

Escherichia coli ItaT toxin reportedly acetylates the α-amino group of the aminoacyl-moiety of Ile-tRNAIle specifically, using acetyl-CoA as an acetyl donor, thereby inhibiting protein synthesis. The mechanism of the substrate specificity of ItaT had remained elusive. Here, we present functional and structural analyses of E. coli ItaT, which revealed the mechanism of ItaT recognition of specific aminoacyl-tRNAs for acetylation. In addition to Ile-tRNAIle, aminoacyl-tRNAs charged with hydrophobic residues, such as Val-tRNAVal and Met-tRNAMet, were acetylated by ItaT in vivo. Ile-tRNAIle, Val-tRNAVal and Met-tRNAMet were acetylated by ItaT in vitro, while aminoacyl-tRNAs charged with other hydrophobic residues, such as Ala-tRNAAla, Leu-tRNALeu and Phe-tRNAPhe, were less efficiently acetylated. A comparison of the structures of E. coli ItaT and the protein N-terminal acetyltransferase identified the hydrophobic residues in ItaT that possibly interact with the aminoacyl moiety of aminoacyl-tRNAs. Mutations of the hydrophobic residues of ItaT reduced the acetylation activity of ItaT toward Ile-tRNAIlein vitro, as well as the ItaT toxicity in vivo. Altogether, the size and shape of the hydrophobic pocket of ItaT are suitable for the accommodation of the specific aminoacyl-moieties of aminoacyl-tRNAs, and ItaT has broader specificity toward aminoacyl-tRNAs charged with certain hydrophobic amino acids.

The RNA-binding protein QKI-7 recruits the poly(A) polymerase GLD-2 for 3' adenylation and selective stabilization of microRNA-122.

MicroRNA-122 (miR-122) is highly expressed in hepatocytes, where it plays an important role in regulating cholesterol and fatty acid metabolism, and it is also a host factor required for hepatitis C virus replication. miR-122 is selectively stabilized by 3' adenylation mediated by the cytoplasmic poly(A) polymerase GLD-2 (also known as PAPD4 or TENT2). However, it is unclear how GLD-2 specifically stabilizes miR-122. Here, we show that QKI7 KH domain-containing RNA binding (QKI-7), one of three isoforms of the QKI proteins, which are members of the signal transduction and activation of RNA (STAR) family of RNA-binding proteins, is involved in miR-122 stabilization. QKI down-regulation specifically decreased the steady-state level of mature miR-122, but did not affect the pre-miR-122 level. We also found that QKI-7 uses its C-terminal region to interact with GLD-2 and its QUA2 domain to associate with the RNA-induced silencing complex protein Argonaute 2 (Ago2), indicating that the GLD-2-QKI-7 interaction recruits GLD-2 to Ago2. QKI-7 exhibited specific affinity to miR-122 and significantly promoted GLD-2-mediated 3' adenylation of miR-122 in vitro Taken together, our findings indicate that miR-122 binds Ago2-interacting QKI-7, which recruits GLD-2 for 3' adenylation and stabilization of miR-122.

Correction: Mitochondrial 16S rRNA Is Methylated by tRNA Methyltransferase TRMT61B in All Vertebrates.

[This corrects the article DOI: 10.1371/journal.pbio.1002557.].

Mitochondrial 16S rRNA Is Methylated by tRNA Methyltransferase TRMT61B in All Vertebrates.

The mitochondrial ribosome, which translates all mitochondrial DNA (mtDNA)-encoded proteins, should be tightly regulated pre- and post-transcriptionally. Recently, we found RNA-DNA differences (RDDs) at human mitochondrial 16S (large) rRNA position 947 that were indicative of post-transcriptional modification. Here, we show that these 16S rRNA RDDs result from a 1-methyladenosine (m1A) modification introduced by TRMT61B, thus being the first vertebrate methyltransferase that modifies both tRNA and rRNAs. m1A947 is conserved in humans and all vertebrates having adenine at the corresponding mtDNA position (90% of vertebrates). However, this mtDNA base is a thymine in 10% of the vertebrates and a guanine in the 23S rRNA of 95% of bacteria, suggesting alternative evolutionary solutions. m1A, uridine, or guanine may stabilize the local structure of mitochondrial and bacterial ribosomes. Experimental assessment of genome-edited Escherichia coli showed that unmodified adenine caused impaired protein synthesis and growth. Our findings revealed a conserved mechanism of rRNA modification that has been selected instead of DNA mutations to enable proper mitochondrial ribosome function.

Molecular basis of glycyl-tRNAGly acetylation by TacT from Salmonella Typhimurium.

Bacterial toxin-antitoxin modules contribute to the stress adaptation, persistence, and dormancy of bacteria for survival under environmental stresses and are involved in bacterial pathogenesis. In Salmonella Typhimurium, the Gcn5-related N-acetyltransferase toxin TacT reportedly acetylates the α-amino groups of the aminoacyl moieties of several aminoacyl-tRNAs, inhibits protein synthesis, and promotes persister formation during the infection of macrophages. Here, we show that TacT exclusively acetylates Gly-tRNAGlyin vivo and in vitro. The crystal structure of the TacT:acetyl-Gly-tRNAGly complex and the biochemical analysis reveal that TacT specifically recognizes the discriminator U73 and G71 in tRNAGly, a combination that is only found in tRNAGly isoacceptors, and discriminates tRNAGly from other tRNA species. Thus, TacT is a Gly-tRNAGly-specific acetyltransferase toxin. The molecular basis of the specific aminoacyl-tRNA acetylation by TacT provides advanced information for the design of drugs targeting Salmonella.

Mechanism of aminoacyl-tRNA acetylation by an aminoacyl-tRNA acetyltransferase AtaT from enterohemorrhagic E. coli.

Toxin-antitoxin systems in bacteria contribute to stress adaptation, dormancy, and persistence. AtaT, a type-II toxin in enterohemorrhagic E. coli, reportedly acetylates the α-amino group of the aminoacyl-moiety of initiator Met-tRNAfMet, thus inhibiting translation initiation. Here, we show that AtaT has a broader specificity for aminoacyl-tRNAs than initially claimed. AtaT efficiently acetylates Gly-tRNAGly, Trp-tRNATrp, Tyr-tRNATyr and Phe-tRNAPhe isoacceptors, in addition to Met-tRNAfMet, and inhibits global translation. AtaT interacts with the acceptor stem of tRNAfMet, and the consecutive G-C pairs in the bottom-half of the acceptor stem are required for acetylation. Consistently, tRNAGly, tRNATrp, tRNATyr and tRNAPhe also possess consecutive G-C base-pairs in the bottom halves of their acceptor stems. Furthermore, misaminoacylated valyl-tRNAfMet and isoleucyl-tRNAfMet are not acetylated by AtaT. Therefore, the substrate selection by AtaT is governed by the specific acceptor stem sequence and the properties of the aminoacyl-moiety of aminoacyl-tRNAs.

Complete chemical structures of human mitochondrial tRNAs.

Mitochondria generate most cellular energy via oxidative phosphorylation. Twenty-two species of mitochondrial (mt-)tRNAs encoded in mtDNA translate essential subunits of the respiratory chain complexes. mt-tRNAs contain post-transcriptional modifications introduced by nuclear-encoded tRNA-modifying enzymes. They are required for deciphering genetic code accurately, as well as stabilizing tRNA. Loss of tRNA modifications frequently results in severe pathological consequences. Here, we perform a comprehensive analysis of post-transcriptional modifications of all human mt-tRNAs, including 14 previously-uncharacterized species. In total, we find 18 kinds of RNA modifications at 137 positions (8.7% in 1575 nucleobases) in 22 species of human mt-tRNAs. An up-to-date list of 34 genes responsible for mt-tRNA modifications are provided. We identify two genes required for queuosine (Q) formation in mt-tRNAs. Our results provide insight into the molecular mechanisms underlying the decoding system and could help to elucidate the molecular pathogenesis of human mitochondrial diseases caused by aberrant tRNA modifications.

Crystal Structure of the Enterohemorrhagic Escherichia coli AtaT-AtaR Toxin-Antitoxin Complex.

AtaT-AtaR is an enterohemorrhagic Escherichia coli toxin-antitoxin system that modulates cellular growth under stress conditions. AtaT and AtaR act as a toxin and its repressor, respectively. AtaT is a member of the GNAT family, and the dimeric AtaT acetylates the α-amino group of the aminoacyl moiety of methionyl initiator tRNAfMet, thereby inhibiting translation initiation. The crystallographic analysis of the AtaT-AtaR complex revealed that the AtaT-AtaR proteins form a heterohexameric [AtaT-(AtaR4)-AtaT] complex, where two V-shaped AtaR dimers bridge two AtaT molecules. The N-terminal region of AtaR is required for its dimerization, and the C-terminal region of AtaR interacts with AtaT. The two AtaT molecules are spatially separated in the AtaT-AtaR complex. AtaT alone forms a dimer in solution, which is enzymatically active. The present structure, in which AtaR prevents AtaT from forming an active dimer, reveals the molecular basis of the AtaT toxicity repression by the antitoxin AtaR.

Function and Regulation of Human Terminal Uridylyltransferases.

RNA uridylylation plays a pivotal role in the biogenesis and metabolism of functional RNAs, and regulates cellular gene expression. RNA uridylylation is catalyzed by a subset of proteins from the non-canonical terminal nucleotidyltransferase family. In human, three proteins (TUT1, TUT4, and TUT7) have been shown to exhibit template-independent uridylylation activity at 3'-end of specific RNAs. TUT1 catalyzes oligo-uridylylation of U6 small nuclear (sn) RNA, which catalyzes mRNA splicing. Oligo-uridylylation of U6 snRNA is required for U6 snRNA maturation, U4/U6-di-snRNP formation, and U6 snRNA recycling during mRNA splicing. TUT4 and TUT7 catalyze mono- or oligo-uridylylation of precursor let-7 (pre-let-7). Let-7 RNA is broadly expressed in somatic cells and regulates cellular proliferation and differentiation. Mono-uridylylation of pre-let-7 by TUT4/7 promotes subsequent Dicer processing to up-regulate let-7 biogenesis. Oligo-uridylylation of pre-let-7 by TUT4/7 is dependent on an RNA-binding protein, Lin28. Oligo-uridylylated pre-let-7 is less responsive to processing by Dicer and degraded by an exonuclease DIS3L2. As a result, let-7 expression is repressed. Uridylylation of pre-let-7 depends on the context of the 3'-region of pre-let-7 and cell type. In this review, we focus on the 3' uridylylation of U6 snRNA and pre-let-7, and describe the current understanding of mechanism of activity and regulation of human TUT1 and TUT4/7, based on their crystal structures that have been recently solved.