Position 34 of tRNA is a discriminative element for m5C38 modification by human DNMT2.

Dnmt2, a member of the DNA methyltransferase superfamily, catalyzes the formation of 5-methylcytosine at position 38 in the anticodon loop of tRNAs. Dnmt2 regulates many cellular biological processes, especially the production of tRNA-derived fragments and intergenerational transmission of paternal metabolic disorders to offspring. Moreover, Dnmt2 is closely related to human cancers. The tRNA substrates of mammalian Dnmt2s are mainly detected using bisulfite sequencing; however, we lack supporting biochemical data concerning their substrate specificity or recognition mechanism. Here, we deciphered the tRNA substrates of human DNMT2 (hDNMT2) as tRNAAsp(GUC), tRNAGly(GCC) and tRNAVal(AAC). Intriguingly, for tRNAAsp(GUC) and tRNAGly(GCC), G34 is the discriminator element; whereas for tRNAVal(AAC), the inosine modification at position 34 (I34), which is formed by the ADAT2/3 complex, is the prerequisite for hDNMT2 recognition. We showed that the C32U33(G/I)34N35 (C/U)36A37C38 motif in the anticodon loop, U11:A24 in the D stem, and the correct size of the variable loop are required for Dnmt2 recognition of substrate tRNAs. Furthermore, mammalian Dnmt2s possess a conserved tRNA recognition mechanism.

Unique anticodon loop conformation with the flipped-out wobble nucleotide in the crystal structure of unbound tRNAVal.

During protein synthesis on ribosome, tRNA recognizes its cognate codon of mRNA through base-pairing with the anticodon. The 5'-end nucleotide of the anticodon is capable of wobble base-pairing, offering a molecular basis for codon degeneracy. The wobble nucleotide is often targeted for post-transcriptional modification, which affects the specificity and fidelity of the decoding process. Flipping-out of a wobble nucleotide in the anticodon loop has been proposed to be necessary for modifying enzymes to access the target nucleotide, which has been captured in selective structures of protein-bound complexes. Meanwhile, all other structures of free or ribosome-bound tRNA display anticodon bases arranged in stacked conformation. We report the X-ray crystal structure of unbound tRNAVal1 to a 2.04 Å resolution showing two different conformational states of wobble uridine in the anticodon loop, one stacked on the neighboring base and the other swiveled out toward solvent. In addition, the structure reveals a rare magnesium ion coordination to the nitrogen atom of a nucleobase, which has been sampled very rarely among known structures of nucleic acids.

Structural diversity and phylogenetic distribution of valyl tRNA-like structures in viruses.

Viruses commonly use specifically folded RNA elements that interact with both host and viral proteins to perform functions important for diverse viral processes. Examples are found at the 3' termini of certain positive-sense ssRNA virus genomes where they partially mimic tRNAs, including being aminoacylated by host cell enzymes. Valine-accepting tRNA-like structures (TLSVal) are an example that share some clear homology with canonical tRNAs but have several important structural differences. Although many examples of TLSVal have been identified, we lacked a full understanding of their structural diversity and phylogenetic distribution. To address this, we undertook an in-depth bioinformatic and biochemical investigation of these RNAs, guided by recent high-resolution structures of a TLSVal We cataloged many new examples in plant-infecting viruses but also in unrelated insect-specific viruses. Using biochemical and structural approaches, we verified the secondary structure of representative TLSVal substrates and tested their ability to be valylated, confirming previous observations of structural heterogeneity within this class. In a few cases, large stem-loop structures are inserted within variable regions located in an area of the TLS distal to known host cell factor binding sites. In addition, we identified one virus whose TLS has switched its anticodon away from valine, causing a loss of valylation activity; the implications of this remain unclear. These results refine our understanding of the structural and functional mechanistic details of tRNA mimicry and how this may be used in viral infection.

Broadly applicable oligonucleotide mass spectrometry for the analysis of RNA writers and erasers in vitro.

RNAs are post-transcriptionally modified by dedicated writer or eraser enzymes that add or remove specific modifications, respectively. Mass spectrometry (MS) of RNA is a useful tool to study the modification state of an oligonucleotide (ON) in a sensitive manner. Here, we developed an ion-pairing reagent free chromatography for positive ion detection of ONs by low- and high-resolution MS, which does not interfere with other types of small compound analyses done on the same instrument. We apply ON-MS to determine the ONs from an RNase T1 digest of in vitro transcribed tRNA, which are purified after ribozyme-fusion transcription by automated size exclusion chromatography. The thus produced tRNAValAAC is substrate of the human tRNA ADAT2/3 enzyme and we confirm the deamination of adenosine to inosine and the formation of tRNAValIACin vitro by ON-MS. Furthermore, low resolution ON-MS is used to monitor the demethylation of ONs containing 1-methyladenosine by bacterial AlkB in vitro. The power of high-resolution ON-MS is demonstrated by the detection and mapping of modified ONs from native total tRNA digested with RNase T1. Overall, we present an oligonucleotide MS method which is broadly applicable to monitor in vitro RNA (de-)modification processes and native RNA.

Detection of Inosine on Transfer RNAs without a Reverse Transcription Reaction.

Inosine at the "wobble" position (I34) is one of the few essential posttranscriptional modifications in tRNAs (tRNAs). It results from the deamination of adenosine and occurs in bacteria on tRNAArgACG and in eukarya on six or seven additional tRNA substrates. Because inosine is structurally a guanosine analogue, reverse transcriptases recognize it as a guanosine. Most methods used to examine the presence of inosine rely on this phenomenon and detect the modified base as a change in the DNA sequence that results from the reverse transcription reaction. These methods, however, cannot always be applied to tRNAs because reverse transcription can be compromised by the presence of other posttranscriptional modifications. Here we present SL-ID (splinted ligation-based inosine detection), a reverse transcription-free method for detecting inosine based on an I34-dependent specific cleavage of tRNAs by endonuclease V, followed by a splinted ligation and polyacrylamide gel electrophoresis analysis. We show that the method can detect I34 on different tRNA substrates and can be applied to total RNA derived from different species, cell types, and tissues. Here we apply the method to solve previous controversies regarding the modification status of mammalian tRNAArgACG.

A tRNA-derived fragment competes with mRNA for ribosome binding and regulates translation during stress.

Posttranscriptional processing of RNA molecules is a common strategy to enlarge the structural and functional repertoire of RNomes observed in all 3 domains of life. Fragmentation of RNA molecules of basically all functional classes has been reported to yield smaller non-protein coding RNAs (ncRNAs) that typically possess different roles compared with their parental transcripts. Here we show that a valine tRNA-derived fragment (Val-tRF) that is produced under certain stress conditions in the halophilic archaeon Haloferax volcanii is capable of binding to the small ribosomal subunit. As a consequence of Val-tRF binding mRNA is displaced from the initiation complex which results in global translation attenuation in vivo and in vitro. The fact that the archaeal Val-tRF also inhibits eukaryal as well as bacterial protein biosynthesis implies a functionally conserved mode of action. While tRFs and tRNA halves have been amply identified in recent RNA-seq project, Val-tRF described herein represents one of the first functionally characterized tRNA processing products to date.

Stabilization of eukaryotic ribosomal termination complexes by deacylated tRNA.

Stabilization of the ribosomal complexes plays an important role in translational control. Mechanisms of ribosome stabilization have been studied in detail for initiation and elongation of eukaryotic translation, but almost nothing is known about stabilization of eukaryotic termination ribosomal complexes. Here, we present one of the mechanisms of fine-tuning of the translation termination process in eukaryotes. We show that certain deacylated tRNAs, remaining in the E site of the ribosome at the end of the elongation cycle, increase the stability of the termination and posttermination complexes. Moreover, only the part of eRF1 recognizing the stop codon is stabilized in the A site of the ribosome, and the stabilization is not dependent on the hydrolysis of peptidyl-tRNA. The determinants, defining this property of the tRNA, reside in the acceptor stem. It was demonstrated by site-directed mutagenesis of tRNA(Val) and construction of a mini-helix structure identical to the acceptor stem of tRNA. The mechanism of this stabilization is different from the fixation of the unrotated state of the ribosome by CCA end of tRNA or by cycloheximide in the E site. Our data allow to reveal the possible functions of the isodecoder tRNAs in eukaryotes.

Structure of the large ribosomal subunit from human mitochondria.

Human mitochondrial ribosomes are highly divergent from all other known ribosomes and are specialized to exclusively translate membrane proteins. They are linked with hereditary mitochondrial diseases and are often the unintended targets of various clinically useful antibiotics. Using single-particle cryogenic electron microscopy, we have determined the structure of its large subunit to 3.4 angstrom resolution, revealing 48 proteins, 21 of which are specific to mitochondria. The structure unveils an adaptation of the exit tunnel for hydrophobic nascent peptides, extensive remodeling of the central protuberance, including recruitment of mitochondrial valine transfer RNA (tRNA(Val)) to play an integral structural role, and changes in the tRNA binding sites related to the unusual characteristics of mitochondrial tRNAs.

Unexpected expansion of tRNA substrate recognition by the yeast m1G9 methyltransferase Trm10.

N-1 Methylation of the nearly invariant purine residue found at position 9 of tRNA is a nucleotide modification found in multiple tRNA species throughout Eukarya and Archaea. First discovered in Saccharomyces cerevisiae, the tRNA methyltransferase Trm10 is a highly conserved protein both necessary and sufficient to catalyze all known instances of m1G9 modification in yeast. Although there are 19 unique tRNA species that contain a G at position 9 in yeast, and whose fully modified sequence is known, only 9 of these tRNA species are modified with m1G9 in wild-type cells. The elements that allow Trm10 to distinguish between structurally similar tRNA species are not known, and sequences that are shared between all substrate or all nonsubstrate tRNAs have not been identified. Here, we demonstrate that the in vitro methylation activity of yeast Trm10 is not sufficient to explain the observed pattern of modification in vivo, as additional tRNA species are substrates for Trm10 m1G9 methyltransferase activity. Similarly, overexpression of Trm10 in yeast yields m1G9 containing tRNA species that are ordinarily unmodified in vivo. Thus, yeast Trm10 has a significantly broader tRNA substrate specificity than is suggested by the observed pattern of modification in wild-type yeast. These results may shed light onto the suggested involvement of Trm10 in other pathways in other organisms, particularly in higher eukaryotes that contain up to three different genes with sequence similarity to the single TRM10 gene in yeast, and where these other enzymes have been implicated in pathways beyond tRNA processing.

Determining RNA three-dimensional structures using low-resolution data.

Knowing the 3-D structure of an RNA is fundamental to understand its biological function. Nowadays X-ray crystallography and NMR spectroscopy are systematically applied to newly discovered RNAs. However, the application of these high-resolution techniques is not always possible, and thus scientists must turn to lower resolution alternatives. Here, we introduce a pipeline to systematically generate atomic resolution 3-D structures that are consistent with low-resolution data sets. We compare and evaluate the discriminative power of a number of low-resolution experimental techniques to reproduce the structure of the Escherichia coli tRNA(VAL) and P4-P6 domain of the Tetrahymena thermophila group I intron. We test single and combinations of the most accessible low-resolution techniques, i.e. hydroxyl radical footprinting (OH), methidiumpropyl-EDTA (MPE), multiplexed hydroxyl radical cleavage (MOHCA), and small-angle X-ray scattering (SAXS). We show that OH-derived constraints are accurate to discriminate structures at the atomic level, whereas EDTA-based constraints apply to global shape determination. We provide a guide for choosing which experimental techniques or combination of thereof is best in which context. The pipeline represents an important step towards high-throughput low-resolution RNA structure determination.

Deoxyribozyme-based, semisynthetic access to stable peptidyl-tRNAs exemplified by tRNAVal carrying a macrolide antibiotic resistance peptide.

We present a protocol for the reliable synthesis of non-hydrolyzable 3'-peptidyl-tRNAs that contain all the respective genuine nucleoside modifications. The approach is exemplified by tRNA(Val)-3'-NH-VFLVM-NH(2) and relies on commercially available Escherichia coli tRNA(Val). This tRNA was cleaved site-specifically within the TΨC loop using a 10-23 type DNA enzyme to obtain a 58 nt tRNA 5'-fragment which contained the modifications. After cleavage of the 2',3'-cyclophosphate moiety from the 5'-fragment, it was ligated to the 18 nt RNA-pentapeptide conjugate which had been chemically synthesized. By this methodology, tRNA(Val)-3'-NH-VFLVM-NH(2) is accessible in efficient manner. Furthermore, we point out that the approach is applicable to other types of tRNA.

tRNA-derived fragments target the ribosome and function as regulatory non-coding RNA in Haloferax volcanii.

Nonprotein coding RNA (ncRNA) molecules have been recognized recently as major contributors to regulatory networks in controlling gene expression in a highly efficient manner. These RNAs either originate from their individual transcription units or are processing products from longer precursor RNAs. For example, tRNA-derived fragments (tRFs) have been identified in all domains of life and represent a growing, yet functionally poorly understood, class of ncRNA candidates. Here we present evidence that tRFs from the halophilic archaeon Haloferax volcanii directly bind to ribosomes. In the presented genomic screen of the ribosome-associated RNome, a 26-residue-long fragment originating from the 5' part of valine tRNA was by far the most abundant tRF. The Val-tRF is processed in a stress-dependent manner and was found to primarily target the small ribosomal subunit in vitro and in vivo. As a consequence of ribosome binding, Val-tRF reduces protein synthesis by interfering with peptidyl transferase activity. Therefore this tRF functions as ribosome-bound small ncRNA capable of regulating gene expression in H. volcanii under environmental stress conditions probably by fine tuning the rate of protein production.

A novel mitochondrial tRNA(Val) T1658C mutation identified in a CPEO family.

PURPOSE: To analyze mitochondrial DNA (mt DNA) gene mutations in a 19-year-old female patient, who presented with chronic progressive external ophthalmoplegia (CPEO), together with her mother and younger sister. METHODS: The diagnosis of mitochondrial myopathy was made based on clinical and biologic analysis. Histochemical methods were used to detect ragged-red fibers (RRFs) and ragged-blue fibers (RBFs) on a muscle biopsy of the patient. All mitochondrial gene DNA fragments of the patient, her mother, and younger sister were amplified by polymerase chain reaction. The products were sequenced and compared with reference databases. RESULTS: A novel T1658C mutation and a known A10006G mutation were identified in the mitochondrial tRNA(Val) gene and the tRNA(Gly) gene, respectively, in the patient, her mother, and younger sister. The T1658C mutation changes the T loop structure of mitochondrial tRNA(Val) and the A10006G mutation disturbs the D loop of mitochondrial tRNA(Gly). CONCLUSIONS: The T1658C and A10006G mutations of mtDNA may be responsible for the pathogenesis of the patient with CPEO.

Longitudinal-relaxation-enhanced NMR experiments for the study of nucleic acids in solution.

Atomic-resolution information on the structure and dynamics of nucleic acids is essential for a better understanding of the mechanistic basis of many cellular processes. NMR spectroscopy is a powerful method for studying the structure and dynamics of nucleic acids; however, solution NMR studies are currently limited to relatively small nucleic acids at high concentrations. Thus, technological and methodological improvements that increase the experimental sensitivity and spectral resolution of NMR spectroscopy are required for studies of larger nucleic acids or protein-nucleic acid complexes. Here we introduce a series of imino-proton-detected NMR experiments that yield an over 2-fold increase in sensitivity compared to conventional pulse schemes. These methods can be applied to the detection of base pair interactions, RNA-ligand titration experiments, measurement of residual dipolar (15)N-(1)H couplings, and direct measurements of conformational transitions. These NMR experiments employ longitudinal spin relaxation enhancement techniques that have proven useful in protein NMR spectroscopy. The performance of these new experiments is demonstrated for a 10 kDa TAR-TAR*(GA) RNA kissing complex and a 26 kDa tRNA.

Measurement of imino 1H-1H residual dipolar couplings in RNA.

Imino (1)H-(15)N residual dipolar couplings (RDCs) provide additional structural information that complements standard (1)H-(1)H NOEs leading to improvements in both the local and global structure of RNAs. Here, we report measurement of imino (1)H-(1)H RDCs for the Iron Responsive Element (IRE) RNA and native E. coli tRNA(Val) using a BEST-Jcomp-HMQC2 experiment. (1)H-(1)H RDCs are observed between the imino protons in G-U wobble base pairs and between imino protons on neighboring base pairs in both RNAs. These imino (1)H-(1)H RDCs complement standard (1)H-(15)N RDCs because the (1)H-(1)H vectors generally point along the helical axis, roughly perpendicular to (1)H-(15)N RDCs. The use of longitudinal relaxation enhancement increased the signal-to-noise of the spectra by ~3.5-fold over the standard experiment. The ability to measure imino (1)H-(1)H RDCs offers a new restraint, which can be used in NMR domain orientation and structural studies of RNAs.

Solution structure of tRNAVal from refinement of homology model against residual dipolar coupling and SAXS data.

A procedure is presented for refinement of a homology model of E. coli tRNA(Val), originally based on the X-ray structure of yeast tRNA(Phe), using experimental residual dipolar coupling (RDC) and small angle X-ray scattering (SAXS) data. A spherical sampling algorithm is described for refinement against SAXS data that does not require a globbic approximation, which is particularly important for nucleic acids where such approximations are less appropriate. Substantially higher speed of the algorithm also makes its application favorable for proteins. In addition to the SAXS data, the structure refinement employed a sparse set of NMR data consisting of 24 imino N-H(N) RDCs measured with Pf1 phage alignment, and 20 imino N-H(N) RDCs obtained from magnetic field dependent alignment of tRNA(Val). The refinement strategy aims to largely retain the local geometry of the 58% identical tRNA(Phe) by ensuring that the atomic coordinates for short, overlapping segments of the ribose-phosphate backbone and the conserved base pairs remain close to those of the starting model. Local coordinate restraints are enforced using the non-crystallographic symmetry (NCS) term in the XPLOR-NIH or CNS software package, while still permitting modest movements of adjacent segments. The RDCs mainly drive the relative orientation of the helical arms, whereas the SAXS restraints ensure an overall molecular shape compatible with experimental scattering data. The resulting structure exhibits good cross-validation statistics (jack-knifed Q (free) = 14% for the Pf1 RDCs, compared to 25% for the starting model) and exhibits a larger angle between the two helical arms than observed in the X-ray structure of tRNA(Phe), in agreement with previous NMR-based tRNA(Val) models.

Determining genetic background in captive stocks of cynomolgus macaques (Macaca fascicularis).

BACKGROUND: As in other model organisms, genetic background in the non-human primates Macaca mulatta and Macaca fascicularis is an experimental variable that affects the response of other study variables. Genetic background in model organisms is manipulated by breeding schemes but is generally pre-determined by the source population used to found captive stocks. In M. fascicularis three such sources have been distinguished, however, these are not routinely taken into consideration when designing research. METHODS: We exemplify a mitochondrial DNA (mtDNA)-based strategy to trace the maternal geographic origins of M. fascicularis animals of unspecified origins. RESULTS: Macaca fascicularis of unspecified origins kept at primate research centers carry mtDNA haplotypes representing all three major genetic subdivisions. CONCLUSIONS: We suggest that the genetic background of study animals could be better specified in the future using an mtDNA-based approach, which would enable informed selection of study animals and help reduce variation within and among studies.

Overexpression of human mitochondrial valyl tRNA synthetase can partially restore levels of cognate mt-tRNAVal carrying the pathogenic C25U mutation.

Phenotypic diversity associated with pathogenic mutations of the human mitochondrial genome (mtDNA) has often been explained by unequal segregation of the mutated and wild-type genomes (heteroplasmy). However, this simple hypothesis cannot explain the tissue specificity of disorders caused by homoplasmic mtDNA mutations. We have previously associated a homoplasmic point mutation (1624C>T) in MTTV with a profound metabolic disorder that resulted in the neonatal deaths of numerous siblings. Affected tissues harboured a marked biochemical defect in components of the mitochondrial respiratory chain, presumably due to the extremely low (<1%) steady-state levels of mt-tRNA(Val). In primary myoblasts and transmitochondrial cybrids established from the proband (index case) and offspring, the marked respiratory deficiency was lost and steady-state levels of the mutated mt-tRNA(Val) were greater than in the biopsy material, but were still an order of magnitude lower than in control myoblasts. We present evidence that the generalized decrease in steady-state mt-tRNA(Val) observed in the homoplasmic 1624C>T-cell lines is caused by a rapid degradation of the deacylated form of the abnormal mt-tRNA(Val). By both establishing the identity of the human mitochondrial valyl-tRNA synthetase then inducing its overexpression in transmitochondrial cell lines, we have been able to partially restore steady-state levels of the mutated mt-tRNA(Val), consistent with an increased stability of the charged mt-tRNA. These data indicate that variations in the levels of VARS2L between tissue types and patients could underlie the difference in clinical presentation between individuals homoplasmic for the 1624C>T mutation.

Effects of conserved D/T loop substitutions in the pre-tRNA substrate on tRNase Z catalysis.

tRNAs are transcribed as precursors with a 5' end leader and a 3' end trailer. In the course of tRNA maturation, RNase P removes the 5' end leader and tRNase Z can endonucleolytically remove the 3' end trailer. A domain remote from the active site of tRNase Z recognizes and binds substrate, principally through contacts with the elbow (D/T loops) of the tRNA. To evaluate possible contacts, processing kinetics was performed using human nuclear encoded pre-tRNA(Arg) with substitutions in conserved D and T loop nucleotides. Changes in K(M) observed with some of the substitutions suggest contacts between tRNase Z and substrate tRNA in this region, and changes in tRNA structure provide an additional basis for interpretation of the kinetic effects.

Comparison of alignment tensors generated for native tRNA(Val) using magnetic fields and liquid crystalline media.

Residual dipolar couplings (RDCs) complement standard NOE distance and J-coupling torsion angle data to improve the local and global structure of biomolecules in solution. One powerful application of RDCs is for domain orientation studies, which are especially valuable for structural studies of nucleic acids, where the local structure of a double helix is readily modeled and the orientations of the helical domains can then be determined from RDC data. However, RDCs obtained from only one alignment media generally result in degenerate solutions for the orientation of multiple domains. In protein systems, different alignment media are typically used to eliminate this orientational degeneracy, where the combination of RDCs from two (or more) independent alignment tensors can be used to overcome this degeneracy. It is demonstrated here for native E. coli tRNA(Val) that many of the commonly used liquid crystalline alignment media result in very similar alignment tensors, which do not eliminate the 4-fold degeneracy for orienting the two helical domains in tRNA. The intrinsic magnetic susceptibility anisotropy (MSA) of the nucleobases in tRNA(Val) was also used to obtain RDCs for magnetic alignment at 800 and 900 MHz. While these RDCs yield a different alignment tensor, the specific orientation of this tensor combined with the high rhombicity for the tensors in the liquid crystalline media only eliminates two of the four degenerate orientations for tRNA(Val). Simulations are used to show that, in optimal cases, the combination of RDCs obtained from liquid crystalline medium and MSA-induced alignment can be used to obtain a unique orientation for the two helical domains in tRNA(Val).