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Background and Purpose: Late-onset mitochondrial disorders are diagnostically challenging with significant heterogeneity in disease presentation. A case is reported of a 67-year-old gentleman who presented with a 3-month history of seizures, recurrent encephalopathy, ataxia and weight loss, preceded by recent initiation of haemodialysis for end-stage chronic kidney disease., Methods: Extensive work-up including serological, cerebrospinal fluid, magnetic resonance imaging and electroencephalography was performed. Whole exome sequencing and muscle biopsy confirmed the diagnosis., Results: Magnetic resonance imaging brain demonstrated a single non-enhancing T2 fluid attenuated inversion recovery hyperintense cortical/subcortical signal change in the right temporal lobe and cerebellar atrophy. Given the subacute presentation of uncertain aetiology, he was empirically treated for autoimmune/paraneoplastic encephalitis. Despite radiological resolution of the cortical abnormality 2 weeks later, there was no clinical improvement. Further collateral history unveiled a mildly ataxic gait and longstanding hearing loss suggestive of a genetic cause. Whole exome sequencing revealed a likely pathogenic, heteroplasmic mitochondrial DNA variant in the MT-TV gene, m.1659T>C, present at higher levels of heteroplasmy in muscle (91%) compared to other mitotic tissues. A high fat/protein diet and multivitamins including co-enzyme Q10 were commenced. Treatment of the nutritional deficiency and avoidance of intermittent fasting due to unreliable oral intake secondary to encephalopathy probably contributed to the clinical improvement seen over the ensuing few months, with resolution of his encephalopathy and return to his baseline gait and weight., Conclusion: An adult case is reported with an acute neurological presentation mimicking encephalitis, caused by a heteroplasmic m.1659T>C MT-TV variant, previously reported once in a child who displayed a different clinical phenotype., (© 2024 The Author(s). European Journal of Neurology published by John Wiley & Sons Ltd on behalf of European Academy of Neurology.)

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., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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 tRNA Val1 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., (© 2021 Jeong and Kim; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

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 (TLS Val ) are an example that share some clear homology with canonical tRNAs but have several important structural differences. Although many examples of TLS Val 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 TLS Val 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 TLS Val 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., (© 2021 Sherlock et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

tRNA-derived fragments offer a recently identified group of non-coding single-stranded RNAs that are often as abundant as microRNAs in cancer cells and play important roles in carcinogenesis. However, the biological functions of them in breast cancer are still unclear. Hence, we focused on investigating whether tiRNAs could play a key role in the progression of breast cancer. We have identified 5'-tiRNA Val with significantly low expression in breast cancer tissues. The down-regulation of serum 5'-tiRNA Val was positively correlated with stage progression and lymph node metastasis. Overexpression of 5'-tiRNA Val suppressed cells malignant activities. FZD3 was confirmed to be a direct target of 5'-tiRNA Val in breast cancer. In addition, FZD3, β-Catenin, c-myc and cyclinD1 levels in 5'-tiRNA Val overexpressing cells were downregulated while APC was inversely upregulated. Moreover, 5'-tiRNA Val inhibited the FZD3-mediated Wnt/β-Catenin signaling pathway in breast cancer cells. Finally, 5'-tiRNA Val levels differentiated breast cancer from healthy controls with a sensitivity of 90.0% and specificity of 62.7%. This is the first study to show that 5'-tiRNA Val as a new tumor-suppressor through inhibition of FZD3/Wnt/β-Catenin signaling pathway, which could be as a potential diagnostic biomarker for breast cancer., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Noncanonical translation, and particularly initiation on non-AUG codons, are frequently used by viral and cellular mRNAs during virus infection and disease. The Sindbis virus (SINV) subgenomic mRNA (sgRNA) constitutes a unique model system to analyze the translation of a capped viral mRNA without the participation of several initiation factors. Moreover, sgRNA can initiate translation even when the AUG initiation codon is replaced by other codons. Using SINV replicons, we examined the efficacy of different codons in place of AUG to direct the synthesis of the SINV capsid protein. The substitution of AUG by CUG was particularly efficient in promoting the incorporation of leucine or methionine in similar percentages at the amino terminus of the capsid protein. Additionally, valine could initiate translation when the AUG is replaced by GUG. The ability of sgRNA to initiate translation on non-AUG codons was dependent on the integrity of a downstream stable hairpin (DSH) structure located in the coding region. The structural requirements of this hairpin to signal the initiation site on the sgRNA were examined in detail. Of interest, a virus bearing CUG in place of AUG in the sgRNA was able to infect cells and synthesize significant amounts of capsid protein. This virus infects the human haploid cell line HAP1 and the double knockout variant that lacks eIF2A and eIF2D. Collectively, these findings indicate that leucine-tRNA or valine-tRNA can participate in the initiation of translation of sgRNA by a mechanism dependent on the DSH. This mechanism does not involve the action of eIF2, eIF2A, or eIF2D., (© 2019 Sanz et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Cystoseira is a common brown algal genus widely distributed throughout the Atlantic and Mediterranean regions whose taxonomical assignment of specimens is often hampered by intra- and interspecific morphological variability. In this study, three mitochondrial regions, namely cytochrome oxidase subunit 1 (COI), 23S rDNA (23S), and 23S-tRNAVal intergenic spacer (mt-spacer) were used to analyse the phylogenetic relationships of 22 Cystoseira taxa (n = 93 samples). A total of 135 sequences (48 from COI, 43 from 23S and 44 from mt-spacer) were newly generated and analysed together with Cystoseira sequences (9 COI, 31 23S and 35 mt-spacer) from other authors. Phylogenetic analysis of these three markers identified 3 well-resolved clades and also corroborated the polyphyletic nature of the genus. The resolution of Cystoseira taxa within the three clades improves significantly when the inclusion of specimens of related genera was minimized. COI and mt-spacer markers resolved the phylogeny of some of the Cystoseira taxa, such as the C. baccata, C. foeniculacea and C. usneoides. Furthermore, trends between phylogeny, embryonic development and available chemotaxonomic classifications were identified, showing that phylogenetic, chemical and morphological data should be taken into account to study the evolutionary relationships among the algae currently classified as Cystoseira. The resolution of Cystoseira macroalgae into three well supported clades achieved here is relevant for a more accurate isolation and identification of natural compounds and the implementation of conservation measures for target species., Competing Interests: The authors have declared that no competing interests exist.

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 tRNA Arg ACG 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 tRNA Arg ACG .

We recently identified and described a putative prophage on the genomic island FhaGI-1 located within the genome of Francisella hispaniensis AS02-814 ( F. tularensis subsp. novicida -like 3523). In this study, we constructed two variants of a Francisella phage integration vector, called pFIV1-Val and pFIV2-Val ( Francisella Integration Vector-tRNA Val -specific), using the attL/R- sites and the site-specific integrase (FN3523_1033) of FhaGI-1, a chloramphenicol resistance cassette and a sacB gene for counter selection of transformants against the vector backbone. We inserted the respective sites and genes into vector pUC57-Kana to allow for propagation in Escherichia coli . The constructs generated a circular episomal form in E. coli which could be used to transform Francisella spp . where FIV-Val stably integrated site specifically into the tRNA Val gene of the genome, whereas pUC57-Kana is lost due to counter selection. Functionality of the new vector was demonstrated by the successfully complementation of a Francisella mutant strain. The vectors were stable in vitro and during host-cell infection without selective pressure. Thus, the vectors can be applied as a further genetic tool in Francisella research, expanding the present genetic tools by an integrative element. This new element is suitable to perform long-term experiments with different Francisella species.

We describe the case of a 40-year-old-man with mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) syndrome, with cardiomyopathy and severe heart failure. He had a mitochondrial transfer RNA (tRNA) mutation (m.1616A>G) of the (tRNA-Val) gene, and it was not found in MELAS syndrome ever before. The presence of this newly observed tRNA-Val mutation (m.1616A>G) may induce multiple respiratory chain enzyme deficiencies and contribute to MELAS syndrome symptoms that are associated with mitochondrial DNA (mtDNA) mutations. We report that the pathognomonic symptom in MELAS syndrome caused by this newly observed mtDNA mutation may be rapid progression of cardiomyopathy and severe heart failure., Competing Interests: Competing interests: None declared., (© BMJ Publishing Group Ltd (unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

The number of species recognized in section Asperae of the flowering plant genus Hydrangea differs widely between subsequent revisions. This variation is largely centered around the H. aspera species complex, with numbers of recognized species varying from one to nearly a dozen. Despite indications of molecular variation in this complex, no sequence-based species delimitation methods have been employed to evaluate the primarily morphology-based species boundaries. In the present study, a multi-locus coalescent-based approach to species delimitation is employed in order to identify separate evolutionary lines within H. sect. Asperae, using four chloroplast and four nuclear molecular markers. Eight lineages were recovered within the focal group, of which five correspond with named morphotypes. The other three lineages illustrate types of conflict between molecular species delimitation and traditional morphology-based taxonomy. One molecular lineage comprises two named morphotypes, which possibly diverged recently enough to not have developed sufficient molecular divergence. A second conflict is found in H. strigosa. This morphotype is recovered as a separate lineage when occurring in geographic isolation, but when occurring in sympatry with two other morphotypes (H. aspera and H. robusta), the coalescent species delimitation lumps these taxa into a single putative species., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Ischemic injuries will lead to necrotic tissue damage, and post-ischemia angiogenesis plays critical roles in blood flow restoration and tissue recovery. Recently, several types of small RNAs have been reported to be involved in this process. In this study, we first generated a rat brain ischemic model to investigate the involvement of new types of small RNAs in ischemia. We utilized deep sequencing and bioinformatics analyses to demonstrate that the level of small RNA fragments derived from tRNAs strikingly increased in the ischemic rat brain. Among these sequences, tRNA(Val)- and tRNA(Gly)-derived small RNAs account for the most abundant segments. The up-regulation of tRNA(Val)- and tRNA(Gly)-derived fragments was verified through northern blot and quantitative PCR analyses. The levels of these two fragments also increased in a mouse hindlimb ischemia model and cellular hypoxia model. Importantly, up-regulation of the tRNA(Val)- and tRNA(Gly)-derived fragments in endothelial cells inhibited cell proliferation, migration and tube formation. Furthermore, we showed that these small RNAs are generated by angiogenin cleavage. Our results indicate that tRNA-derived fragments are involved in tissue ischemia, and we demonstrate for the first time that tRNA(Val)- and tRNA(Gly)-derived fragments inhibit angiogenesis by modulating the function of endothelial cells.

Diagnosis of hypertrophic cardiomyopathy (HCM) involved the screening for the candidate pathogenic mitochondrial DNA (mtDNA) mutations. However, a poor genotype to phenotype correction is common. Neutral polymorphisms in mt-tRNA gene are recognized as a potential cause for HCM. Thus, assigning the pathogenicity for mt-tRNA mutation is important for both clinical and genetic scientists when confronted with a disease exhibiting the clinical and biochemical features of mitochondrial dysfunction. In this report, we reassess the role of mt-tRNA(Val) 1628C > T mutation in HCM expression. We first carried out a systematic search in the published database, finding out the genotype and phenotype corrections for this mutation. Moreover, we perform a phylogenetic approach to see whether this mutation is conserved or not. Most strikingly, the 1628C > T mutation is not conserved and a slight change of entropy is observed between the wild type and the mutant carrying the 1628C > T mutation. Our data indicate that the 1628C > T transition should not be regarded as a mutation associated with HCM.

Recently, we identified a putative prophage on a genomic island (GI) within the genome sequence of Francisella hispaniensis isolate AS0-814 (Francisella tularensis subsp. novicida-like 3523) by the analysis of the CRISPR-Cas systems of Francisella. Various spacer DNAs within the CRISPR region of different F. tularensis subsp. novicida strains were found to be homologous to the putative prophage (Schunder et al., 2013, Int. J. Med. Microbiol. 303:51-60). Now we identified the GI (FhaGI-1) as a mobile element which is able to form a circular episomal structure. The circular episomal form of FhaGI-1 is generated by F. hispaniensis, and the excision of the island is an integrase-dependent and site-specific process. Furthermore, we could demonstrate that the excision of the island is also possible in other bacterial species (Escherichia coli). In addition, we could show that a genetically generated small variant of the island is also functional and, after its electroporation into strain F. tularensis subsp. holarctica LVS, the GI was stable and site-specifically integrated into the genome of the transformants. The integrase is sufficient for the integration and excision of the small variant into and from the DNA backbone, respectively. Thus, the element may be suitable to be used as a genetic tool in F. tularensis research. Furthermore, we identified the tRNA(Val) gene of Francisella as an integration site for GIs. Genomic island FphGI-1 was identified in Francisella philomiragia ATCC 25016. We were not able to detect the episomal form of this GI, probably due to a mutated attR site. However, we could demonstrate that integrative GIs are present in Francisella and that they may allow horizontal gene transfer between different Francisella species., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

We assessed phylogenetic and systematic relationships among 17 out of 23 species of Theloderma and all three species of Nyctixalus from 2412bp sequences of the mitochondrial DNA genes of 12S rRNA, tRNA(val), and 16S rRNA using maximum likelihood and Bayesian inference methods. With the exception of T. moloch, Theloderma and Nyctixalus are confirmed to form a clade, in which each genus also forms a clade. Theloderma moloch is phylogenetically outside these clades and closer to samples from Chiromantis, Feihyla, Gracixalus, Kurixalus, Philautus, Polypedates, Raorchestes, and Rhacophorus. Within Theloderma, T. horridum and T. stellatum form the sister taxon to a clade comprising the remaining species. The basal split within the latter clade groups T. asperum, T. licin, T. petilum, and T. ryabovi as the sister to a clade comprising T. bicolor, T. chuyangsinense, T. corticale, T. gordoni, T. laeve, T. lateriticum, T. nebulosum, T. rhododiscus, and T. truongsonense. Our phylogenetic results indicate homoplastic evolution of four morphological characters: small vs. large body size, presence of vomerine teeth, presence of a vocal opening in males, and interdigital webbing on hands. The common ancestor of Theloderma and Nyctixalus is inferred to have arisen in the area including the current Sunda region., (Copyright © 2015 Elsevier Inc. All rights reserved.)

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., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Mutations in mitochondrial tRNA genes are one of the most important causes of mitochondrial diseases. Recently, a novel mt-tRNA(Val) T1658C mutation has been reported to be associated with chronic progressive external ophthalmoplegia (CPEO). To test this association, we performed a phylogenetic analysis of T1658C mutation, moreover, we used the bioinformatic tool to predict the thermodynamic change of tRNA(Val) with and without this mutation. Surprisingly, T1658C mutation was not evolutionary conserved and had little effect on tRNA(Val) folding. These data indicated that T1658C mutation should still be categorized as a polymorphism.

By way of whole-exome sequencing, we identified a homozygous missense mutation in VARS2 in one subject with microcephaly and epilepsy associated with isolated deficiency of the mitochondrial respiratory chain (MRC) complex I and compound heterozygous mutations in TARS2 in two siblings presenting with axial hypotonia and severe psychomotor delay associated with multiple MRC defects. The nucleotide variants segregated within the families, were absent in Single Nucleotide Polymorphism (SNP) databases and are predicted to be deleterious. The amount of VARS2 and TARS2 proteins and valyl-tRNA and threonyl-tRNA levels were decreased in samples of afflicted patients according to the genetic defect. Expression of the corresponding wild-type transcripts in immortalized mutant fibroblasts rescued the biochemical impairment of mitochondrial respiration and yeast modeling of the VARS2 mutation confirmed its pathogenic role. Taken together, these data demonstrate the role of the identified mutations for these mitochondriopathies. Our study reports the first mutations in the VARS2 and TARS2 genes, which encode two mitochondrial aminoacyl-tRNA synthetases, as causes of clinically distinct, early-onset mitochondrial encephalopathies., (© 2014 The Authors. **Human Mutation published by Wiley Periodicals, Inc.)

We describe four patients from three independent families with the m.1644G>A in the MT-TV gene, previously reported without demonstration of its deleterious impact. Very high mutation proportion co-segregated with cytochrome oxidase defect in single muscle fibers and respiratory defect in cybrids as shown by spectrophotometric assays and polarography. The mutation appeared to have a very steep threshold effect with asymptomatic life up to 70% mutation proportion, progressive encephalopathy above 80% and severe Leigh-like syndrome above 95% mutation. One patient did not fit within that frame but presented with characteristics suggesting the presence of an additional disease., (Copyright © 2014 © Elsevier B.V. and Mitochondria Research Society. Published by Elsevier B.V. All rights reserved.)

Here we report that RNase P is required for the initial separation of all seven valine tRNAs from three distinct polycistronic transcripts (valV valW, valU valX valY lysY and lysT valT lysW valZ lysY lysZ lysQ). Particularly significant is the mechanism by which RNase P processes the valU and lysT polycistronic transcripts. Specifically, the enzyme initiates processing by first removing the Rho-independent transcription terminators from the primary valU and lysT transcripts. Subsequently, it proceeds in the 3' → 5' direction generating one pre-tRNA at a time. Based on the absolute requirement for RNase P processing of all three primary transcripts, inactivation of the enzyme leads to a > 4-fold decrease in the levels of both type I and type II valine tRNAs. The ability of RNase P to initiate tRNA processing at the 3' ends of long primary transcripts by endonucleolytically removing the Rho-independent transcription terminator represents a previously unidentified function for the enzyme, which is responsible for generating the mature 5' termini of all 86 E. coli tRNAs. RNase E only plays a very minor role in the processing of all three valine polycistronic transcripts., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Homoplasmic m.1624C>T mutation of the mitochondrial tRNA(Val) gene was previously demonstrated to cause fatal neonatal Leigh syndrome. Here, we report the clinical phenotypes of a Japanese male and his mother with heteroplasmic m.1624C>T mutation. The 36-year-old male presented with repeated episodes of consciousness disturbance since the age of 25, cognitive decline, and personality change. Cerebrospinal fluid levels of lactate and pyruvate were elevated. His mother showed similar symptoms and course. The mutation m.1624C>T was identified heteroplasmically in the proband's muscle and leukocytes and in the mother's leukocytes. The heteroplasmy load decreased with age., (Copyright © 2012 Elsevier B.V. and Mitochondria Research Society. All rights reserved.)

In this study, the phylogenetic relationships among 13 pangasiids and six schilbids of Thailand were reconstructed based on the almost complete mitochondrial cytochrome b (cyt b), 12S rRNA, tRNA-Val and 16S rRNA, as well as the partial nuclear recombination-activating gene 1 (rag1) sequences by using the maximum likelihood and the Bayesian inference methods of phylogenetic reconstruction. The reconstructed phylogeny based on the concatenated sequence data set recovered Pangasiidae and Schilbidae as reciprocally monophyletic groups. Within Pangasiidae, four major clades were recovered, which according to the cyt b genetic distances can be categorized into four genera: Pangasius, Pseudolais, Helicophagus and Pangasianodon. The genus Pangasianodon was strongly supported as the most basal taxon within pangasiids, whereas Pseudolais + Helicophagus were recovered as a sister group of Pangasius. Within the latter, the giant pangasius Pangasius sanitwongsei was recovered as a sister group of the spot pangasius Pangasius larnaudii, Pangasius krempfi as a sister group of Pangasius nasutus + Pangasius conchophilus and Pangasius polyuranodon as a sister species of Pangasius macronema. Other internal phylogenetic relationships, however, were unresolved. Within Schilbidae, Pseudeutropius was supported as the most basal lineage. Eutropiichthys was recovered as a sister group of Clupisoma. The enigmatic Clupisoma sinense was recognized as more closely related to Laides longibarbis than to Clupisoma prateri. Thus, based on the cyt b genetic distances, a recategorization of C. sinense to the genus Laides is suggested. On the basis of a relaxed clock fossil calibration, the divergence of pangasiids and schilbids was estimated to have occurred 14·93 million years before present (b.p.) during the Miocene epoch. The separation between Pangasiidae and Schilbidae took place c. 13·12 Mb.p. during the early middle Miocene. The estimated divergence time of pangasiids is similar to the age of the calibrated fossil, Cetopangasius chaetobranchus, which was discovered in north-central Thailand. This suggests that the oldest pangasiid ancestor diverged into diverse genera in the area., (© 2012 The Authors. Journal of Fish Biology © 2012 The Fisheries Society of the British Isles.)

The aim of this study was to identify the genetic defect in two patients having cardiac dysfunction accompanied by neurological symptoms, and in one case MRI evidence of cortical and cerebellar atrophy with hyperintensities in the basal ganglia. Muscle biopsies from each patient revealed single and combined mitochondrial respiratory chain deficiency. The complete mtDNA sequencing of both patients revealed two transitions in the mitochondrial tRNA(Val) gene (MT-TV) (m.1628C>T in Patient 1, and m.1644G>A in Patient 2). The functional and molecular analyses reported here suggest that the MT-TV gene should be routinely considered in the diagnosis of mitochondrial cardiomyopathies., (Copyright © 2011 Elsevier B.V. and Mitochondria Research Society. All rights reserved. All rights reserved.)

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.

An m.1630A>G mutation in the mitochondrial tRNA(Val) (MTTV) was identified in a patient with hearing impairment, short stature and new onset of stroke. This mutation has previously been identified in a patient with the mitochondrial neurogastrointestinal encephalopathy syndrome (MNGIE). The mother of the proband also had high levels of the m.1630A>G allele present in blood and other tissues, without symptoms. To confirm the pathogenicity of this mutation, we created cybrid cell lines with various mutation loads. The m.1630A>G mutation impairs oxygen consumption, affects the stability of the MTTV and reduces the levels of subunits of the electron transport chain., (© 2011 © Elsevier B.V. and Mitochondria Research Society. All rights reserved.)

Mitochondrial encephalopathy, lactic acidosis and strokelike episodes (MELAS) syndrome is a mitochondrial disorder characterized by a wide variety of clinical presentations and a multisystemic organ involvement. In this study, we report a Tunisian girl with clinical features of MELAS syndrome who was negative for the common m.3243A>G mutation, but also for the reported mitochondrial DNA (mtDNA) mutations and deletions. Screening of the entire mtDNA genome showed several known mitochondrial variants besides to a novel transition m.1640A>G affecting a wobble adenine in the anticodon stem region of the tRNA(Val). This nucleotide was conserved and it was absent in 150 controls suggesting its pathogenicity. In addition, no mutations were found in the nuclear polymerase gamma-1 gene (POLG1). These results suggest further investigation nuclear genes encoding proteins responsible for stability and structural components of the mtDNA or to the oxidative phosphorylation machinery to explain the phenotypic variability in the studied family., (Copyright © 2011 Elsevier Inc. All rights reserved.)

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.

In this study, we infer the phylogenetic relationships within commercial shrimp using sequence data from a novel mitochondrial marker consisting of an approximately 530-bp region of the 16S ribosomal RNA (rRNA)/transfer RNA (tRNA)(Val) genes compared with two other mitochondrial genes: 16S rRNA and cytochrome c oxidase I (COI). All three mitochondrial markers were considerably AT rich, exhibiting values up to 78.2% for the species Penaeus monodon in the 16S rRNA/tRNA(Val) genes, notably higher than the average among other Malacostracan mitochondrial genomes. Unlike the 16S rRNA and COI genes, the 16S rRNA/tRNA(Val) marker evidenced that Parapenaeus is more closely related to Metapenaeus than to Solenocera, a result that seems to be more in agreement with the taxonomic status of these genera. To our knowledge, our study using the 16S rRNA/tRNA(Val) gene as a marker for phylogenetic analysis offers the first genetic evidence to confirm that Pleoticus muelleri and Solenocera agassizi constitute a separate group and that they are more related to each other than to genera belonging to the family Penaeidae. The 16S rRNA/tRNA(Val) region was also found to contain more variable sites (56%) than the other two regions studied (33.4% for the 16S rRNA region and 42.7% for the COI region). The presence of more variable sites in the 16S rRNA/tRNA(Val) marker allowed the interspecific differentiation of all 19 species examined. This is especially useful at the commercial level for the identification of a large number of shrimp species, particularly when the lack of morphological characteristics prevents their differentiation.

Transfer RNA is highly modified. Nucleotide 37 of the anticodon loop is represented by various modified nucleotides. In Escherichia coli, the valine-specific tRNA (cmo(5)UAC) contains a unique modification, N(6)-methyladenosine, at position 37; however, the enzyme responsible for this modification is unknown. Here we demonstrate that the yfiC gene of E. coli encodes an enzyme responsible for the methylation of A37 in tRNA(1)(Val). Inactivation of yfiC gene abolishes m(6)A formation in tRNA(1)(Val), while expression of the yfiC gene from a plasmid restores the modification. Additionally, unmodified tRNA(1)(Val) can be methylated by recombinant YfiC protein in vitro. Although the methylation of m(6)A in tRNA(1)(Val) by YfiC has little influence on the cell growth under standard conditions, the yfiC gene confers a growth advantage under conditions of osmotic and oxidative stress.

While mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is typically associated with mutations in the nuclear gene encoding for thymidine phosphorylase (ECGF1, TYMP), a similar clinical phenotype was described in patients carrying mutations in the nuclear-encoded polymerase gamma (POLG1) as well as a few mitochondrial tRNA genes. Here we report a novel mutation in the mitochondrial tRNA(Val) (MTTV) gene in a girl presenting with clinical symptoms of MNGIE-like gastrointestinal dysmotility and cachexia. Clinical, histological, biochemical and single cell investigations were performed. The heteroplasmic m.1630A>G mutation was detected in the mitochondrial tRNA(Val) (MTTV) gene in the patient's muscle, blood leukocytes and myoblasts, as well as in blood DNA of the unaffected mother. We provide clinical, biochemical, histological, and molecular genetic evidence on the single cell level for the pathogenicity of this mutation. Our finding adds to the genetic heterogeneity of MNGIE-like gastrointestinal symptoms and highlights the importance of a thorough genetic workup in case of suspected mitochondrial disease.

The bases at positions 32 and 38 in the tRNA anticodon loop are known to have a specific conservation depending upon the anticodon triplets. Here we report that evolutionarily conserved pairs of bases at positions 32 and 38 in tRNA(Ala)(GGC) prevent misreading of a near-cognate valine codon, GUC. The tRNA(Ala)(GGC) molecules with the conserved A32-U38 and C32-G38 pairs do not read GUC, whereas those with three representative nonconserved pairs, U32-U38, U32-A38 and C32-A38, direct the misincorporation of alanine at this valine codon into the peptide chain. Overexpression of the nonconserved tRNA(Ala)(GGC) in Escherichia coli is toxic and prevents cell growth. These results suggested that the bases at positions 32 and 38 in tRNA(Ala)(GGC) evolved to preserve the fidelity of the cognate codon reading.

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.

Mitochondrial encephalopathy, lactic acidosis and stroke-like episodes (MELAS) is the most common mitochondrial disease due to mitochondrial DNA (mtDNA) mutations. At least 15 distinct mtDNA mutations have been associated with MELAS, and about 80% of the cases are caused by the A3243G tRNA(Leu(UUR)) gene mutation. We report here a novel tRNA(Val) mutation in a 37-year-old woman with manifestations of MELAS, and compare her clinicopathological phenotype with other rare cases associated tRNA(Val) mutations.

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.

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.

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).

One of the most prevalent base modifications involved in decoding is uridine 5-oxyacetic acid at the wobble position of tRNA. It has been known for several decades that this modification enables a single tRNA to decode all four codons in a degenerate codon box. We have determined structures of an anticodon stem-loop of tRNA(Val) containing the modified uridine with all four valine codons in the decoding site of the 30S ribosomal subunit. An intramolecular hydrogen bond involving the modification helps to prestructure the anticodon loop. We found unusual base pairs with the three noncomplementary codon bases, including a G.U base pair in standard Watson-Crick geometry, which presumably involves an enol form for the uridine. These structures suggest how a modification in the uridine at the wobble position can expand the decoding capability of a tRNA.

Ribozymes are naturally-occurring catalytic RNAs from the viroid world and are being engineered in the laboratory to perform sequence-specific cleavage of a desired mRNA target. Since their Nobel Prize-winning discovery, there has been considerable interest in the utility of ribozymes as gene therapeutic agents to silence disease-causing genes. This technology is not perfect, but extensive efforts to improve upon natural design of ribozymes have enabled these RNA molecules to perform various tasks. In this chapter, we highlight the construction of two types of ribozymes: conventional and hybrid hammerhead ribozymes. The hybrid ribozyme described here is an improved version of the basic hammerhead motif with the following features: (a) the use of the RNA polymerase III (polIII) tRNAVal promoter to achieve a high level of transcription, (b) 5' linkage to the cloverleaf-shaped tRNAVal to enhance intracellular stability and cytoplasmic transport, and (c) a 3' end poly-(A) tail to act as a "molecular anchor" for endogenous RNA helicases endowing the ribozyme ability to disentangle higher-order structures of the target mRNA. Randomized hybrid ribozyme libraries have been used successfully for revelation of gene functions involved in metastasis, invasion, differentiation, apoptosis, endoplasmic reticulum stress and may be extended to gene functions involved in innate or induced cellular senescence of human cells.

Here, we provide an exemplar-approach phylogeny of the xystodesmid millipede tribe Apheloriini with a focus on genus-group relationships-particularly of the genus Brachoria. Exemplars for the phylogenetic analysis were chosen to represent the maximum breadth of morphological diversity within all nominal genera in the tribe Apheloriini, and to broadly sample the genus Brachoria. In addition, three closely related tribes were used (Rhysodesmini, Nannariini, and Pachydesmini). Morphological and DNA sequence data were scored for Bayesian inference of phylogeny. Phylogenetic analysis resulted in polyphyletic genera Brachoria and Sigmoria, a monophyletic Apheloriini, and a "southern clade" that contains most of the tribal species diversity. We used this phylogeny to track morphological character histories and reconstruct ancestral states using stochastic character mapping. Based on the findings from the character mapping study, the diagnostic feature of the genus Brachoria, the cingulum, evolved independently in two lineages. We compared our phylogeny against prior classifications using Bayes factor hypothesis-testing and found that our phylogenetic hypothesis is inconsistent with the previous hypotheses underlying the most recent classification. With our preferred total-evidence phylogeny as a framework for taxonomic modifications, we describe a new genus, Appalachioria; supply phylogenetic diagnoses of monophyletic taxa; and provide a phylogeny-based classification for the tribe Apheloriini.

Coordinated translocation of the tRNA-mRNA complex by the ribosome occurs in a precise, stepwise movement corresponding to a distance of three nucleotides along the mRNA. Frameshift suppressor tRNAs generally contain an extra nucleotide in the anticodon loop and they subvert the normal mechanisms used by the ribosome for frame maintenance. The mechanism by which suppressor tRNAs traverse the ribosome during translocation is poorly understood. Here, we demonstrate translocation of a tRNA by four nucleotides from the A site to the P site, and from the P site to the E site. We show that translocation of a punctuated mRNA is possible with an extra, unpaired nucleotide between codons. Interestingly, the NMR structure of the four nucleotide anticodon stem-loop reveals a conformation different from the canonical tRNA structure. Flexibility within the loop may allow conformational adjustment upon A site binding and for interacting with the four nucleotide codon in order to shift the mRNA reading frame.

We present a phylogeny of the Neotropical genus Engystomops (= Physalaemus pustulosus species group) based on sequences of approximately 2.4 kb of mtDNA, (12S rRNA, valine-tRNA, and 16S rRNA) and propose a phylogenetic nomenclature. The phylogeny includes all described taxa and two unnamed species. All analyses indicate that Engystomops is monophyletic and contains two basal allopatric clades. Clade I (Edentulus) includes E. pustulosus and the Amazonian E. petersi + E. cf. freibergi. Clade II (Duovox) includes all species distributed in W Ecuador and NW Peru. Brevivox, a clade of small-sized species is strongly supported within Duovox. Populations of Engystomops pustulosus fall into two well-supported clades, each of which occupies two disjunct portions of the species range. Overall, our phylogeny is congruent with most previous hypotheses. This study is among the few published species-level phylogenies of Neotropical amphibians derived from molecular datasets. A review of the proportion of new species detected by similar studies suggests that the increasing use of molecular techniques will lead to the discovery of a vast number of species of Neotropical amphibians.

Objective: To screen efficient siRNA for inhibiting hepatitis B virus using the technique of PCR-based tRNA(val) Pol III-shRNA expression cassettes (SECs)., Methods: Based on core gene sequence of HBV, five target sites of siRNA were designed. tRNAval Pol III-shRNA expression cassettes produced by one-step overlapping extension PCR strategy were co-transfected with HBV C gene and pC-EGFP plasmid into AD293 cells respectively. Forty-eight hours after transfection, fluorescence of HBVC-GFP protein was detected by fluorescence-activated cell sorting (FACS); HBV C mRNA was detected by semi-quantitative RT-PCR. HBV-producing HepG2. 2. 15 cells were transfected with selected SECs for 72 h, HBsAg and HBeAg in the cell culture medium were detected by radioimmunoassay assay (RIA). HBV pgRNA from cell total RNA was detected by semi-quantitative PCR., Result: Co-transfection with pC-GFP plasmid and SECs into AD293 cells resulted in inhibition expression of HBV C gene and decrease of EGFP fluorescence intensity. SEC-492i showed most significant inhibition effect on HBV C-EGFP expression compared with other SECs. Selected SEC-492i or SEC-282i targeting core gene could efficiently decrease expression of HBeAg and the level of HBV pgRNA in a dose-dependent manner. SEC-492i inhibited HBV replication and antigen expression in a more efficient way than SEC-282i at the same final concentration., Conclusion: The expressed shRNA, which targets sites on HBV C mRNA in 492i, is to have having most efficient RNAi effect. tRNAval Pol III-shRNA expression cassettes produced by one-step overlapping extension PCR strategy should be useful for identification of optimal siRNA.

Stigmatella aurantiaca displays a complex developmental life cycle in response to starvation conditions that results in the formation of tree-like fruiting bodies capable of producing spores. The phage Mx8, first isolated from the close relative Myxococcus xanthus, is unable to infect S. aurantiaca cells and integrate into the genome. However, plasmids containing Mx8 fragments encoding the integrase and attP are able to integrate at the attB locus in the S. aurantiaca genome by site-specific recombination. After recombination between attP and attB, the S. aurantiaca cells were incapable of building normal fruiting bodies but formed clumps and fungus-like structures characteristic of intermediate stages of development displayed by the wild type. We identified two tRNA genes, trnD and trnV, encoding tRNA(Asp) and tRNA(Val), respectively, composing an operon at the attB locus of S. aurantiaca. Integration of attP-containing plasmids resulted in the incorporation of the t(Mx8) terminator sequence, in addition to a short sequence of Mx8 DNA downstream of trnD. The integrant was unable to process the trnD transcript at the normal 3' processing site and displayed a lower level of expression of the trnVD operon. In addition, several developmentally regulated proteins were no longer produced in mutants following insertion at the attB locus. We hypothesize that the integration of the t(Mx8) terminator sequence results in reduced levels of mature tRNA(Asp) and tRNA(Val) and that altered protein production during development is thereby responsible for the observed phenotype. The trnVD locus thus defines a new developmental checkpoint for Stigmatella aurantiaca.

The biological role of many nonessential tRNA modifications outside of the anticodon remains elusive despite their evolutionary conservation. We show here that m7G46 methyltransferase Trm8p/Trm82p acts as a hub of synthetic interactions with several tRNA modification enzymes, resulting in temperature-sensitive growth. Analysis of three double mutants indicates reduced levels of tRNA(Val(AAC)), consistent with a role of the corresponding modifications in maintenance of tRNA levels. Detailed examination of a trm8-delta trm4-delta double mutant demonstrates rapid degradation of preexisting tRNA(Val(AAC)) accompanied by its de-aminoacylation. Multiple copies of tRNA(Val(AAC)) suppress the trm8-delta trm4-delta growth defect, directly implicating this tRNA in the phenotype. These results define a rapid tRNA degradation (RTD) pathway that is independent of the TRF4/RRP6-dependent nuclear surveillance pathway. The degradation of an endogenous tRNA species at a rate typical of mRNA decay demonstrates a critical role of nonessential modifications for tRNA stability and cell survival.

Cardinalfishes of the genus Apogon (Apogonidae) are one of the most speciose (>200 species) and numerically dominant fishes in coral reefs. Although the genus is divided into 10 subgenera, more than 70% of the species are included in the subgenus Ostorhinchus, most having either horizontal or vertical lines on the body. The phylogenetic relationship among 32 species of subgenus Ostorhinchus and 11 species of four other subgenera of Apogon, based on mitochondrially encoded 12S and 16S ribosomal genes and intervening tRNA(Val) gene, were investigated, using two species of the apogonid genus Fowleria as outgroups. The analyses demonstrated that Ostorhinchus (the most speciose subgenus) was polyphyletic, comprising at least three lineages, Ostorhinchus I, II, and III. Ostorhinchus I included two species, A. (O.) amboinensis and A. (O.) sangiensis, being a sister group to subgenus Zoramia. Ostorhinchus II and III included species with horizontal and vertical lines on the body, respectively. The respective monophylies of the latter two groups, together with a molecular clock calibration, indicated that in the evolutionary history of the genus, basic stripe patterns evolved first (more than 20 million years BP), with subsequent pattern diversification and modification.

To downregulate expression of the beta1 integrin subunit in endothelial cells, plasmid encoding the ribozyme cassette containing hammerhead ribozyme flanked at the 5' terminus by tRNA(Val) and at the 3' terminus by constitutive transport element sequences was constructed. When used to transfect immortalized human endothelial cell line EA.hy 926, it selectively blocked the synthesis of the beta1 integrin subunit and thus inhibited migration and proliferation of the cells. Thus, this construct may be a valuable tool to control the proangiogenic phenotype of stimulated endothelial cells.

Aminoacyl-tRNA synthetases are responsible for attaching amino acid residues to the tRNA 3'-end. The two classes of synthetases approach tRNA as mirror images, with opposite but symmetrical stereochemistries that allow the class I enzymes to attach amino acid residues to the 2'-hydroxyl group of the terminal ribose, whereas, the class II enzymes attach amino acid residues to the 3'-hydroxyl group. However, we show here that the attachment of cysteine to tRNA(Cys) by the class I cysteinyl-tRNA synthetase (CysRS) is flexible; the enzyme is capable of using either the 2' or 3'-hydroxyl group as the attachment site. The molecular basis for this flexibility was investigated. Introduction of the nucleotide U73 of tRNA(Cys) into tRNA(Val) was found to confer the flexibility. While valylation of the wild-type tRNA(Val) by the class I ValRS was strictly dependent on the terminal 2'-hydroxyl group, that of the U73 mutant of tRNA(Val) occurred at either the 2' or 3'-hydroxyl group. Thus, the single nucleotide U73 of tRNA has the ability to break the stereo barrier of amino acid attachment to tRNA, by mobilizing the 2' and 3'-hydroxyl groups of A76 in flexible geometry with respect to the tRNA acceptor stem.

The effects of post-transcriptional modifications on the structure and dynamics of Escherichia coli tRNA(val) were studied by NMR spectroscopy. NMR chemical shift data and residual dipolar couplings were used to show that the local secondary and tertiary structures are very similar in native and unmodified tRNA(val). Rigid body restrained molecular dynamics calculations showed that the global structure of tRNA is unchanged by the post-transcriptional modifications. Deuterium exchange NMR experiments were used to probe the dynamics and flexibility of native and unmodified tRNA(val). A similar set of slowly exchanging (t(1/2) > 3 min) imino protons were observed in both tRNAs, but the rates of exchange for the slowest exchanging imino protons were approximately 20 times faster in unmodified than in native tRNA. These results demonstrate that the dynamics and flexibility of tRNA(val), but not the local or global structure, are significantly affected by post-transcriptional modifications.

Objective: To examine mitochondrial DNA mutations in mitochondrial myopathy., Methods: Three suspected cases of mitochondrial myopathy were examined by HE staining, histochemical staining methods and electron microscopy. The mutations in all 22 tRNA genes of mitochondrial genome were screened by polymerase chain reaction-single strand conformation polymorphism and DNA sequencing., Results: The three cases were diagnosed as mitochondrial myopathy. The examinations revealed that patient 1 had a homoplasmic A1627G mutation in tRNA-Val gene, and patient 2 had a heteroplasmic A1627G/A mutation in tRNA-Val gene, and patient 3 had two mutationsuone was homoplasmic T5554C mutation in tRNA-Trp gene, the other was heteroplasmic A10412C/A mutation in tRNA-Arg gene., Conclusion: tRNA genes mutations of mtDNA might be one of the etiologies of mitochondrial myopathy.