Mechanism of U6 snRNA oligouridylation by human TUT1.

U6 snRNA is a catalytic RNA responsible for pre-mRNA splicing reactions and undergoes various post-transcriptional modifications during its maturation process. The 3'-oligouridylation of U6 snRNA by the terminal uridylyltransferase, TUT1, provides the Lsm-binding site in U6 snRNA for U4/U6 di-snRNP formation and this ensures pre-mRNA splicing. Here, we present the crystal structure of human TUT1 (hTUT1) complexed with U6 snRNA, representing the post-uridylation of U6 snRNA by hTUT1. The N-terminal ZF-RRM and catalytic palm clamp the single-stranded AUA motif between the 5'-short stem and the 3'-telestem of U6 snRNA, and the ZF-RRM specifically recognizes the AUA motif. The ZF and the fingers hold the telestem, and the 3'-end of U6 snRNA is placed in the catalytic pocket of the palm for oligouridylation. The oligouridylation of U6 snRNA depends on the internal four-adenosine tract in the 5'-part of the telestem of U6 snRNA, and hTUT1 adds uridines until the internal adenosine tract can form base-pairs with the 3'-oligouridine tract. Together, the recognition of the specific structure and sequence of U6 snRNA by the multi-domain TUT1 protein and the intrinsic sequence and structure of U6 snRNA ensure the oligouridylation of U6 snRNA.

Mechanism of tRNA recognition by heterotetrameric glycyl-tRNA synthetase from lactic acid bacteria.

Glycyl-tRNA synthetases (GlyRSs) have different oligomeric structures depending on the organisms. While a dimeric α2 GlyRS species is present in archaea, eukaryotes and some eubacteria, a heterotetrameric α2ß2 GlyRS species is found in most eubacteria. Here, we present the crystal structure of heterotetrameric α2ß2 GlyRS, consisting of the full-length α and ß subunits, from Lactobacillus plantarum (LpGlyRS), gram-positive lactic bacteria. The α2ß2LpGlyRS adopts the same X-shaped structure as the recently reported Escherichia coli α2ß2 GlyRS. A tRNA docking model onto LpGlyRS suggests that the α and ß subunits of LpGlyRS together recognize the L-shaped tRNA structure. The α and ß subunits of LpGlyRS together interact with the 3'-end and the acceptor region of tRNAGly, and the C-terminal domain of the ß subunit interacts with the anticodon region of tRNAGly. The biochemical analysis using tRNA variants showed that in addition to the previously defined determinants G1C72 and C2G71 base pairs, C35, C36 and U73 in eubacterial tRNAGly, the identification of bases at positions 4 and 69 in tRNAGly is required for efficient glycylation by LpGlyRS. In this case, the combination of a purine base at Position 4 and a pyrimidine base at Position 69 in tRNAGly is preferred.

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 m6A modification of U6 snRNA by human METTL16.

The N6-methyladenosine modification at position 43 (m6A43) of U6 snRNA is catalyzed by METTL16, and is important for the 5'-splice site recognition by U6 snRNA during pre-mRNA splicing. Human METTL16 consists of the N-terminal methyltransferase domain (MTD) and the C-terminal vertebrate conserved region (VCR). While the MTD has an intrinsic property to recognize a specific sequence in the distinct structural context of RNA, the VCR functions have remained uncharacterized. Here, we present structural and functional analyses of the human METTL16 VCR. The VCR increases the affinity of METTL16 toward U6 snRNA, and the conserved basic region in VCR is important for the METTL16-U6 snRNA interaction. The VCR structure is topologically homologous to the C-terminal RNA binding domain, KA1, in U6 snRNA-specific terminal uridylyl transferase 1 (TUT1). A chimera of the N-terminal MTD of METTL16 and the C-terminal KA1 of TUT1 methylated U6 snRNA more efficiently than the MTD, indicating the functional conservation of the VCR and KA1 for U6 snRNA biogenesis. The VCR interacts with the internal stem-loop (ISL) within U6 snRNA, and this interaction would induce the conformational rearrangement of the A43-containing region of U6 snRNA, thereby modifying the RNA structure to become suitable for productive catalysis by the MTD. Therefore, the MTD and VCR in METTL16 cooperatively facilitate the m6A43 U6 snRNA modification.

In vitro yeast reconstituted translation system reveals function of eIF5A for synthesis of long polypeptide.

We have recently developed an in vitro yeast reconstituted translation system, which is capable of synthesizing long polypeptides. Utilizing the system, we examined the role of eIF5A and its hypusine modification in translating polyproline sequence within long open reading frames. We found that polyproline motif inserted at the internal position of the protein arrests translation exclusively at low Mg2+ concentrations, and peptidylpolyproline-tRNA intrinsically destabilizes 80S ribosomes. We demonstrate that unmodified eIF5A essentially resolves such ribosome stalling; however, the hypusine modification drastically stimulates ability of eIF5A to rescue polyproline-mediated ribosome stalling and is particularly important for the efficient translation of the N-terminal or long internal polyproline motifs.

Crystal structure of human cytoplasmic tRNAHis-specific 5'-monomethylphosphate capping enzyme.

BCDIN3 domain containing RNA methyltransferase, BCDIN3D, monomethylates the 5'-monophosphate of cytoplasmic tRNAHis with a G-1:A73 mispair at the top of an eight-nucleotide-long acceptor helix, using S-adenosyl-l-methionine (SAM) as a methyl group donor. In humans, BCDIN3D overexpression is associated with the tumorigenic phenotype and poor prognosis in breast cancer. Here, we present the crystal structure of human BCDIN3D complexed with S-adenosyl-l-homocysteine. BCDIN3D adopts a classical Rossmann-fold methyltransferase structure. A comparison of the structure with that of the closely related methylphosphate capping enzyme, MePCE, which monomethylates the 5'-γ-phosphate of 7SK RNA, revealed the important residues for monomethyl transfer from SAM onto the 5'-monophosphate of tRNAHis and for tRNAHis recognition by BCDIN3D. A structural model of tRNAHis docking onto BCDIN3D suggested the molecular mechanism underlying the different activities between BCDIN3D and MePCE. A loop in BCDIN3D is shorter, as compared to the corresponding region that forms an α-helix to recognize the 5'-end of RNA in MePCE, and the G-1:A73 mispair in tRNAHis allows the N-terminal α-helix of BCDIN3D to wedge the G-1:A73 mispair of tRNAHis. As a result, the 5'-monophosphate of G-1 of tRNAHis is deep in the catalytic pocket for 5'-phosphate methylation. Thus, BCDIN3D is a tRNAHis-specific 5'-monomethylphosphate capping enzyme that discriminates tRNAHis from other tRNA species, and the structural information presented in this study also provides the molecular basis for the development of drugs against breast cancers.

Human BCDIN3D Is a Cytoplasmic tRNAHis-Specific 5'-Monophosphate Methyltransferase.

Bicoid interacting 3 domain containing RNA methyltransferase (BCDIN3D) is a member of the Bin3 methyltransferase family and is evolutionary conserved from worm to human. BCDIN3D is overexpressed in breast cancer, which is associated with poor prognosis of breast cancers. However, the biological functions and properties of BCDIN3D have been enigmatic. Recent studies have revealed that human BCDIN3D monomethylates 5'-monophsosphate of cytoplasmic tRNAHisin vivo and in vitro. BCDIN3D recognizes the unique and exceptional structural features of cytoplasmic tRNAHis and discriminates tRNAHis from other cytoplasmic tRNA species. Thus, BCDIN3D is a tRNAHis-specific 5'-monophosphate methyltransferase. Methylation of the 5'-phosphate group of tRNAHis does not significantly affect tRNAHis aminoacylation by histidyl-tRNA synthetase in vitro nor the steady state level or stability of tRNAHisin vivo. Hence, methylation of the 5'-phosphate group of tRNAHis by BCDIN3D or tRNAHis itself may be involved in certain unknown biological processes, beyond protein synthesis. This review discusses recent reports on BCDIN3D and the possible association between 5'-phosphate monomethylation of tRNAHis and the tumorigenic phenotype of breast cancer.

Acetate-dependent tRNA acetylation required for decoding fidelity in protein synthesis.

Modification of tRNA anticodons plays a critical role in ensuring accurate translation. N4-acetylcytidine (ac4C) is present at the anticodon first position (position 34) of bacterial elongator tRNAMet. Herein, we identified Bacillus subtilis ylbM (renamed tmcAL) as a novel gene responsible for ac4C34 formation. Unlike general acetyltransferases that use acetyl-CoA, TmcAL activates an acetate ion to form acetyladenylate and then catalyzes ac4C34 formation through a mechanism similar to tRNA aminoacylation. The crystal structure of TmcAL with an ATP analog reveals the molecular basis of ac4C34 formation. The ΔtmcAL strain displayed a cold-sensitive phenotype and a strong genetic interaction with tilS that encodes the enzyme responsible for synthesizing lysidine (L) at position 34 of tRNAIle to facilitate AUA decoding. Mistranslation of the AUA codon as Met in the ΔtmcAL strain upon tilS repression suggests that ac4C34 modification of tRNAMet and L34 modification of tRNAIle act cooperatively to prevent misdecoding of the AUA codon.

Human BCDIN3D monomethylates cytoplasmic histidine transfer RNA.

Human RNA methyltransferase BCDIN3D is overexpressed in breast cancer cells, and is related to the tumorigenic phenotype and poor prognosis of breast cancer. Here, we show that cytoplasmic tRNAHis is the primary target of BCDIN3D in human cells. Recombinant human BCDIN3D, expressed in Escherichia coli, monomethylates the 5΄-monophosphate of cytoplasmic tRNAHis efficiently in vitro. In BCDN3D-knockout cells, established by CRISPR/Cas9 editing, the methyl moiety at the 5΄-monophosphate of cytoplasmic tRNAHis is lost, and the exogenous expression of BCDIN3D in the knockout cells restores the modification in cytoplasmic tRNAHis. BCIDN3D recognizes the 5΄-guanosine nucleoside at position -1 (G-1) and the eight-nucleotide acceptor helix with the G-1-A73 mis-pair at the top of the acceptor stem of cytoplasmic tRNAHis, which are exceptional structural features among cytoplasmic tRNA species. While the monomethylation of the 5΄-monophosphate of cytoplasmic tRNAHis affects neither the overall aminoacylation process in vitro nor the steady-state level of cytoplasmic tRNAHisin vivo, it protects the cytoplasmic tRNAHis transcript from degradation in vitro. Thus, BCDIN3D acts as a cytoplasmic tRNAHis-specific 5΄-methylphosphate capping enzyme. The present results also suggest the possible involvement of the monomethylation of the 5΄-monophosphate of cytoplasmic tRNAHis and/or cytoplasmic tRNAHis itself in the tumorigenesis of breast cancer cells.

The Sam68 nuclear body is composed of two RNase-sensitive substructures joined by the adaptor HNRNPL.

The mammalian cell nucleus contains membraneless suborganelles referred to as nuclear bodies (NBs). Some NBs are formed with an architectural RNA (arcRNA) as the structural core. Here, we searched for new NBs that are built on unidentified arcRNAs by screening for ribonuclease (RNase)-sensitive NBs using 32,651 fluorescently tagged human cDNA clones. We identified 32 tagged proteins that required RNA for their localization in distinct nuclear foci. Among them, seven RNA-binding proteins commonly localized in the Sam68 nuclear body (SNB), which was disrupted by RNase treatment. Knockdown of each SNB protein revealed that SNBs are composed of two distinct RNase-sensitive substructures. One substructure is present as a distinct NB, termed the DBC1 body, in certain conditions, and the more dynamic substructure including Sam68 joins to form the intact SNB. HNRNPL acts as the adaptor to combine the two substructures and form the intact SNB through the interaction of two sets of RNA recognition motifs with the putative arcRNAs in the respective substructures.

Measurement of Acceptor-TΨC Helix Length of tRNA for Terminal A76-Addition by A-Adding Enzyme.

The 3'-terminal CCA (C74C75A76-3') of tRNA is required for protein synthesis. In Aquifex aeolicus, the CCA-3' is synthesized by CC-adding and A-adding enzymes, although in most organisms, CCA is synthesized by a single CCA-adding enzyme. The mechanisms by which the A-adding enzyme adds only A76, but not C74C75, onto tRNA remained elusive. The complex structures of the enzyme with various tRNAs revealed the presence of a single tRNA binding site on the enzyme, with the enzyme measuring the acceptor-TΨC helix length of tRNA. The 3'-C75 of tRNA lacking A76 can reach the active site and the size and shape of the nucleotide binding pocket at the insertion stage are suitable for ATP. The 3'-C74 of tRNA lacking C75A76 cannot reach the active site, although CTP or ATP can bind the active pocket. Thus, the A-adding enzyme adds only A76, but not C74C75, onto tRNA.

Mechanism for template-independent terminal adenylation activity of Qß replicase.

The genomic RNA of Qß virus is replicated by Qß replicase, a template-dependent RNA polymerase complex. Qß replicase has an intrinsic template-independent RNA 3'-adenylation activity, which is required for efficient viral RNA amplification in the host cells. However, the mechanism of the template-independent 3'-adenylation of RNAs by Qß replicase has remained elusive. We determined the structure of a complex that includes Qß replicase, a template RNA, a growing RNA complementary to the template RNA, and ATP. The structure represents the terminal stage of RNA polymerization and reveals that the shape and size of the nucleotide-binding pocket becomes available for ATP accommodation after the 3'-penultimate template-dependent C-addition. The stacking interaction between the ATP and the neighboring Watson-Crick base pair, between the 5'-G in the template and the 3'-C in the growing RNA, contributes to the nucleotide specificity. Thus, the template for the template-independent 3'-adenylation by Qß replicase is the RNA and protein ribonucleoprotein complex.

Mechanism for the alteration of the substrate specificities of template-independent RNA polymerases.

PolyA polymerase (PAP) adds a polyA tail onto the 3'-end of RNAs without a nucleic acid template, using adenosine-5'-triphosphate (ATP) as a substrate. The mechanism for the substrate selection by eubacterial PAP remains obscure. Structural and biochemical studies of Escherichia coli PAP (EcPAP) revealed that the shape and size of the nucleobase-interacting pocket of EcPAP are maintained by an intra-molecular hydrogen-network, making it suitable for the accommodation of only ATP, using a single amino acid, Arg(197). The pocket structure is sustained by interactions between the catalytic domain and the RNA-binding domain. EcPAP has a flexible basic C-terminal region that contributes to optimal RNA translocation for processive adenosine 5'-monophosphate (AMP) incorporations onto the 3'-end of RNAs. A comparison of the EcPAP structure with those of other template-independent RNA polymerases suggests that structural changes of domain(s) outside the conserved catalytic core domain altered the substrate specificities of the template-independent RNA polymerases.

Mechanism for the definition of elongation and termination by the class II CCA-adding enzyme.

The CCA-adding enzyme synthesizes the CCA sequence at the 3' end of tRNA without a nucleic acid template. The crystal structures of class II Thermotoga maritima CCA-adding enzyme and its complexes with CTP or ATP were determined. The structure-based replacement of both the catalytic heads and nucleobase-interacting neck domains of the phylogenetically closely related Aquifex aeolicus A-adding enzyme by the corresponding domains of the T. maritima CCA-adding enzyme allowed the A-adding enzyme to add CCA in vivo and in vitro. However, the replacement of only the catalytic head domain did not allow the A-adding enzyme to add CCA, and the enzyme exhibited (A, C)-adding activity. We identified the region in the neck domain that prevents (A, C)-adding activity and defines the number of nucleotide incorporations and the specificity for correct CCA addition. We also identified the region in the head domain that defines the terminal A addition after CC addition. The results collectively suggest that, in the class II CCA-adding enzyme, the head and neck domains collaboratively and dynamically define the number of nucleotide additions and the specificity of nucleotide selection.

Conserved cysteine residues of GidA are essential for biogenesis of 5-carboxymethylaminomethyluridine at tRNA anticodon.

The 5-carboxymethylaminomethyl modification of uridine (cmnm(5)U) at the anticodon first position occurs in tRNAs that read split codon boxes ending with purine. This modification is crucial for correct translation, by restricting codon-anticodon wobbling. Two conserved enzymes, GidA and MnmE, participate in the cmnm(5)U modification process. Here we determined the crystal structure of Aquifex aeolicus GidA at 2.3 A resolution. The structure revealed the tight interaction of GidA with FAD. Structure-based mutation analyses allowed us to identify two conserved Cys residues in the vicinity of the FAD-binding site that are essential for the cmnm(5)U modification in vivo. Together with mutational analysis of MnmE, we propose a mechanism for the cmnm(5)U modification process where GidA, but not MnmE, attacks the C6 atom of uridine by a mechanism analogous to that of thymidylate synthase. We also present a tRNA-docking model that provides structural insights into the tRNA recognition mechanism for efficient modification.

Molecular basis for maintenance of fidelity during the CCA-adding reaction by a CCA-adding enzyme.

CCA-adding enzyme builds the 3'-end CCA of tRNA without a nucleic acid template. The mechanism for the maintenance of fidelity during the CCA-adding reaction remains elusive. Here, we present almost a dozen complex structures of the class I CCA-adding enzyme and tRNA mini-helices (mini-D(73)N(74), mini-D(73)N(74)C(75) and mini-D(73)C(74)N(75); D(73) is a discriminator nucleotide and N is either A, G, or U). The mini-D(73)N(74) complexes adopt catalytically inactive open forms, and CTP shifts the enzymes to the active closed forms and allows N(74) to flip for CMP incorporation. In contrast, unlike the catalytically active closed form of the mini-D(73)C(74)C(75) complex, the mini-D(73)N(74)C(75) and mini-D(73)C(74)N(75) complexes adopt inactive open forms. Only the mini-D(73)C(74)U(75) accepts AMP to a similar extent as mini-D(73)C(74)C(75), and ATP shifts the enzyme to a closed, active form and allows U(75) to flip for AMP incorporation. These findings suggest that the 3'-region of RNA is proofread, after two nucleotide additions, in the closed, active form of the complex at the AMP incorporation stage. This proofreading is a prerequisite for the maintenance of fidelity for complete CCA synthesis.

Protein-based peptide-bond formation by aminoacyl-tRNA protein transferase.

Eubacterial leucyl/phenylalanyl-tRNA protein transferase (LF-transferase) catalyses peptide-bond formation by using Leu-tRNA(Leu) (or Phe-tRNA(Phe)) and an amino-terminal Arg (or Lys) of a protein, as donor and acceptor substrates, respectively. However, the catalytic mechanism of peptide-bond formation by LF-transferase remained obscure. Here we determine the structures of complexes of LF-transferase and phenylalanyl adenosine, with and without a short peptide bearing an N-terminal Arg. Combining the two separate structures into one structure as well as mutation studies reveal the mechanism for peptide-bond formation by LF-transferase. The electron relay from Asp 186 to Gln 188 helps Gln 188 to attract a proton from the alpha-amino group of the N-terminal Arg of the acceptor peptide. This generates the attacking nucleophile for the carbonyl carbon of the aminoacyl bond of the aminoacyl-tRNA, thus facilitating peptide-bond formation. The protein-based mechanism for peptide-bond formation by LF-transferase is similar to the reverse reaction of the acylation step observed in the peptide hydrolysis reaction by serine proteases.

Complete crystallographic analysis of the dynamics of CCA sequence addition.

CCA-adding polymerase matures the essential 3'-CCA terminus of transfer RNA without any nucleic-acid template. However, it remains unclear how the correct nucleotide triphosphate is selected in each reaction step and how the polymerization is driven by the protein and RNA dynamics. Here we present complete sequential snapshots of six complex structures of CCA-adding enzyme and four distinct RNA substrates with and without CTP (cytosine triphosphate) or ATP (adenosine triphosphate). The CCA-lacking RNA stem extends by one base pair to force the discriminator nucleoside into the active-site pocket, and then tracks back after incorporation of the first cytosine monophosphate (CMP). Accommodation of the second CTP clamps the catalytic cleft, inducing a reorientation of the turn, which flips C74 to allow CMP to be accepted. In contrast, after the second CMP is added, the polymerase and RNA primer are locked in the closed state, which directs the subsequent A addition. Between the CTP- and ATP-binding stages, the side-chain conformation of Arg 224 changes markedly; this is controlled by the global motion of the enzyme and position of the primer terminus, and is likely to achieve the CTP/ATP discrimination, depending on the polymerization stage. Throughout the CCA-adding reaction, the enzyme tail domain firmly anchors the TPsiC-loop of the tRNA, which ensures accurate polymerization and termination.