Publications by authors named "Ya-Ming Hou"

111 Publications

Twice exploration of tRNA +1 frameshifting in an elongation cycle of protein synthesis.

Nucleic Acids Res 2021 Sep;49(17):10046-10060

Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA 19107, USA.

Inducing tRNA +1 frameshifting to read a quadruplet codon has the potential to incorporate a non-natural amino acid into the polypeptide chain. While this strategy is being considered for genome expansion in biotechnology and bioengineering endeavors, a major limitation is a lack of understanding of where the shift occurs in an elongation cycle of protein synthesis. Here, we use the high-efficiency +1-frameshifting SufB2 tRNA, containing an extra nucleotide in the anticodon loop, to address this question. Physical and kinetic measurements of the ribosome reading frame of SufB2 identify twice exploration of +1 frameshifting in one elongation cycle, with the major fraction making the shift during translocation from the aminoacyl-tRNA binding (A) site to the peptidyl-tRNA binding (P) site and the remaining fraction making the shift within the P site upon occupancy of the A site in the +1-frame. We demonstrate that the twice exploration of +1 frameshifting occurs during active protein synthesis and that each exploration is consistent with ribosomal conformational dynamics that permits changes of the reading frame. This work indicates that the ribosome itself is a determinant of changes of the reading frame and reveals a mechanistic parallel of +1 frameshifting with -1 frameshifting.
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http://dx.doi.org/10.1093/nar/gkab734DOI Listing
September 2021

Loss of -methylation of G37 in tRNA induces ribosome stalling and reprograms gene expression.

Elife 2021 08 12;10. Epub 2021 Aug 12.

Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, United States.

-methylation of G37 is required for a subset of tRNAs to maintain the translational reading-frame. While loss of mG37 increases ribosomal +1 frameshifting, whether it incurs additional translational defects is unknown. Here, we address this question by applying ribosome profiling to gain a genome-wide view of the effects of mG37 deficiency on protein synthesis. Using as a model, we show that mG37 deficiency induces ribosome stalling at codons that are normally translated by mG37-containing tRNAs. Stalling occurs during decoding of affected codons at the ribosomal A site, indicating a distinct mechanism than that of +1 frameshifting, which occurs after the affected codons leave the A site. Enzyme- and cell-based assays show that mG37 deficiency reduces tRNA aminoacylation and in some cases peptide-bond formation. We observe changes of gene expression in mG37 deficiency similar to those in the stringent response that is typically induced by deficiency of amino acids. This work demonstrates a previously unrecognized function of mG37 that emphasizes its role throughout the entire elongation cycle of protein synthesis, providing new insight into its essentiality for bacterial growth and survival.
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http://dx.doi.org/10.7554/eLife.70619DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8384417PMC
August 2021

Structural basis for +1 ribosomal frameshifting during EF-G-catalyzed translocation.

Nat Commun 2021 07 30;12(1):4644. Epub 2021 Jul 30.

RNA Therapeutics Institute, Department of Biochemistry and Molecular Pharmacology, UMass Medical School, Worcester, MA, USA.

Frameshifting of mRNA during translation provides a strategy to expand the coding repertoire of cells and viruses. How and where in the elongation cycle +1-frameshifting occurs remains poorly understood. We describe seven ~3.5-Å-resolution cryo-EM structures of 70S ribosome complexes, allowing visualization of elongation and translocation by the GTPase elongation factor G (EF-G). Four structures with a + 1-frameshifting-prone mRNA reveal that frameshifting takes place during translocation of tRNA and mRNA. Prior to EF-G binding, the pre-translocation complex features an in-frame tRNA-mRNA pairing in the A site. In the partially translocated structure with EF-G•GDPCP, the tRNA shifts to the +1-frame near the P site, rendering the freed mRNA base to bulge between the P and E sites and to stack on the 16S rRNA nucleotide G926. The ribosome remains frameshifted in the nearly post-translocation state. Our findings demonstrate that the ribosome and EF-G cooperate to induce +1 frameshifting during tRNA-mRNA translocation.
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http://dx.doi.org/10.1038/s41467-021-24911-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8324841PMC
July 2021

A stress-free strategy to correct point mutations in patient iPS cells.

Stem Cell Res 2021 05 8;53:102332. Epub 2021 Apr 8.

Department of Neuroscience Vickie & Jack Farber Institute for Neuroscience, Sidney Kimmel Medical College, Thomas Jefferson University, 900 Walnut Street, JHN Suite 461, Philadelphia, PA 19107, USA. Electronic address:

When studying patient specific induced pluripotent stem cells (iPS cells) as a disease model, the ideal control is an isogenic line that has corrected the point mutation, instead of iPS cells from siblings or other healthy subjects. However, repairing a point mutation in iPS cells even with the newly developed CRISPR-Cas9 technique remains difficult and time-consuming. Here we report a strategy that makes the Cas9 "knock-in" methodology both hassle-free and error-free. Instead of selecting a Cas9 recognition site close to the point mutation, we chose a site located in the nearest intron. We constructed a donor template with the fragment containing the corrected point mutation as one of the homologous recombination arms flanking a PGK-Puro cassette. After selection with puromycin, positive clones were identified and further transfected with a CRE vector to remove the PGK-Puro cassette. Using this methodology, we successfully repaired the point mutation G2019S of the LRRK2 gene in a Parkinson Disease (PD) patient iPS line and the point mutation R329H of the AARS1 gene in a Charcot-Marie-Tooth disease (CMT) patient iPS line. These isogenic iPS lines are ideal as a control in future studies.
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http://dx.doi.org/10.1016/j.scr.2021.102332DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8283763PMC
May 2021

Insights into genome recoding from the mechanism of a classic +1-frameshifting tRNA.

Nat Commun 2021 01 12;12(1):328. Epub 2021 Jan 12.

Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA, 19107, USA.

While genome recoding using quadruplet codons to incorporate non-proteinogenic amino acids is attractive for biotechnology and bioengineering purposes, the mechanism through which such codons are translated is poorly understood. Here we investigate translation of quadruplet codons by a +1-frameshifting tRNA, SufB2, that contains an extra nucleotide in its anticodon loop. Natural post-transcriptional modification of SufB2 in cells prevents it from frameshifting using a quadruplet-pairing mechanism such that it preferentially employs a triplet-slippage mechanism. We show that SufB2 uses triplet anticodon-codon pairing in the 0-frame to initially decode the quadruplet codon, but subsequently shifts to the +1-frame during tRNA-mRNA translocation. SufB2 frameshifting involves perturbation of an essential ribosome conformational change that facilitates tRNA-mRNA movements at a late stage of the translocation reaction. Our results provide a molecular mechanism for SufB2-induced +1 frameshifting and suggest that engineering of a specific ribosome conformational change can improve the efficiency of genome recoding.
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http://dx.doi.org/10.1038/s41467-020-20373-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7803779PMC
January 2021

Purification and Use of tRNA for Enzymatic Post-translational Addition of Amino Acids to Proteins.

STAR Protoc 2020 Dec 9;1(3):100207. Epub 2020 Dec 9.

Department of Biomedical Sciences, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.

Post-translational addition of amino acids to proteins by enzymes using aminoacyl-tRNA is an emerging regulatory mechanism. Examples include Arg transfer in eukaryotes, Leu/Phe transfer in bacteria, and tRNA-synthetase-mediated addition of amino acids to Lys side chains. Here, we present a method of purification and use of tRNA for such reactions, focusing on tRNA and its use for arginylation. This method can also be used for other tRNA-mediated reactions. For complete details on the use and execution of this protocol, please refer to Avcilar-Kucukgoze et al. (2020).
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http://dx.doi.org/10.1016/j.xpro.2020.100207DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7757669PMC
December 2020

A Label-Free Assay for Aminoacylation of tRNA.

Genes (Basel) 2020 10 7;11(10). Epub 2020 Oct 7.

Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA 19107, USA.

Aminoacylation of tRNA generates an aminoacyl-tRNA (aa-tRNA) that is active for protein synthesis on the ribosome. Quantification of aminoacylation of tRNA is critical to understand the mechanism of specificity and the flux of the aa-tRNA into the protein synthesis machinery, which determines the rate of cell growth. Traditional assays for the quantification of tRNA aminoacylation involve radioactivity, either with a radioactive amino acid or with a [3'-P]-labeled tRNA. We describe here a label-free assay that monitors aminoacylation by biotinylation-streptavidin (SA) conjugation to the α-amine or the α-imine of the aminoacyl group on the aa-tRNA. The conjugated aa-tRNA product is readily separated from the unreacted tRNA by a denaturing polyacrylamide gel, allowing for quantitative measurement of aminoacylation. This label-free assay is applicable to a wide range of amino acids and tRNA sequences and to both classes of aminoacylation. It is more sensitive and robust than the assay with a radioactive amino acid and has the potential to explore a wider range of tRNA than the assay with a [3'-P]-labeled tRNA. This label-free assay reports kinetic parameters of aminoacylation quantitatively similar to those reported by using a radioactive amino acid, suggesting its broad applicability to research relevant to human health and disease.
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http://dx.doi.org/10.3390/genes11101173DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7601589PMC
October 2020

Mg-Dependent Methyl Transfer by a Knotted Protein: A Molecular Dynamics Simulation and Quantum Mechanics Study.

ACS Catal 2020 Aug 22;10(15):8058-8068. Epub 2020 Jun 22.

Centre of New Technologies, University of Warsaw, Warsaw 02-097, Poland.

Mg is required for the catalytic activity of TrmD, a bacteria-specific methyltransferase that is made up of a protein topological knot-fold, to synthesize methylated mG37-tRNA to support life. However, neither the location of Mg in the structure of TrmD nor its role in the catalytic mechanism is known. Using molecular dynamics (MD) simulations, we identify a plausible Mg binding pocket within the active site of the enzyme, wherein the ion is coordinated by two aspartates and a glutamate. In this position, Mg additionally interacts with the carboxylate of a methyl donor cofactor S-adenosylmethionine (SAM). The computational results are validated by experimental mutation studies, which demonstrate the importance of the Mg-binding residues for the catalytic activity. The presence of Mg in the binding pocket induces SAM to adopt a unique bent shape required for the methyl transfer activity and causes a structural reorganization of the active site. Quantum mechanical calculations show that the methyl transfer is energetically feasible only when Mg is bound in the position revealed by the MD simulations, demonstrating that its function is to align the active site residues within the topological knot-fold in a geometry optimal for catalysis. The obtained insights provide the opportunity for developing a strategy of antibacterial drug discovery based on targeting of Mg-binding to TrmD.
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http://dx.doi.org/10.1021/acscatal.0c00059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7462349PMC
August 2020

A Mitochondrial tRNA Mutation Causes Axonal CMT in a Large Venezuelan Family.

Ann Neurol 2020 10 29;88(4):830-842. Epub 2020 Aug 29.

Department of Neurology, University of California, San Francisco, CA, USA.

Objective: The objective of this study was to identify the genetic cause for progressive peripheral nerve disease in a Venezuelan family. Despite the growing list of genes associated with Charcot-Marie-Tooth disease, many patients with axonal forms lack a genetic diagnosis.

Methods: A pedigree was constructed, based on family clinical data. Next-generation sequencing of mitochondrial DNA (mtDNA) was performed for 6 affected family members. Muscle biopsies from 4 family members were used for analysis of muscle histology and ultrastructure, mtDNA sequencing, and RNA quantification. Ultrastructural studies were performed on sensory nerve biopsies from 2 affected family members.

Results: Electrodiagnostic testing showed a motor and sensory axonal polyneuropathy. Pedigree analysis revealed inheritance only through the maternal line, consistent with mitochondrial transmission. Sequencing of mtDNA identified a mutation in the mitochondrial tRNA (mt-tRNA ) gene, m.1661A>G, present at nearly 100% heteroplasmy, which disrupts a Watson-Crick base pair in the T-stem-loop. Muscle biopsies showed chronic denervation/reinnervation changes, whereas biochemical analysis of electron transport chain (ETC) enzyme activities showed reduction in multiple ETC complexes. Northern blots from skeletal muscle total RNA showed severe reduction in abundance of mt-tRNA , and mildly increased mt-tRNA , in subjects compared with unrelated age- and sex-matched controls. Nerve biopsies from 2 affected family members demonstrated ultrastructural mitochondrial abnormalities (hyperplasia, hypertrophy, and crystalline arrays) consistent with a mitochondrial neuropathy.

Conclusion: We identify a previously unreported cause of Charcot-Marie-Tooth (CMT) disease, a mutation in the mt-tRNA , in a Venezuelan family. This work expands the list of CMT-associated genes from protein-coding genes to a mitochondrial tRNA gene. ANN NEUROL 2020;88:830-842.
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http://dx.doi.org/10.1002/ana.25854DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7781299PMC
October 2020

Deacetylation of HSD17B10 by SIRT3 regulates cell growth and cell resistance under oxidative and starvation stresses.

Cell Death Dis 2020 07 23;11(7):563. Epub 2020 Jul 23.

Department of Medical Genetics, Center for Medical Genetics, Peking University Health Science Center, Beijing, 100191, China.

17-beta-hydroxysteroid dehydrogenase 10 (HSD17B10) plays an important role in mitochondrial fatty acid metabolism and is also involved in mitochondrial tRNA maturation. HSD17B10 missense mutations cause HSD10 mitochondrial disease (HSD10MD). HSD17B10 with mutations identified from cases of HSD10MD show loss of function in dehydrogenase activity and mitochondrial tRNA maturation, resulting in mitochondrial dysfunction. It has also been implicated to play roles in the development of Alzheimer disease (AD) and tumorigenesis. Here, we found that HSD17B10 is a new substrate of NAD-dependent deacetylase Sirtuin 3 (SIRT3). HSD17B10 is acetylated at lysine residues K79, K99 and K105 by the acetyltransferase CBP, and the acetylation is reversed by SIRT3. HSD17B10 acetylation regulates its enzymatic activity and the formation of mitochondrial RNase P. Furthermore, HSD17B10 acetylation regulates the intracellular functions, affecting cell growth and cell resistance in response to stresses. Our results demonstrated that acetylation is an important regulation mechanism for HSD17B10 and may provide insight into interrupting the development of AD.
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http://dx.doi.org/10.1038/s41419-020-02763-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7378191PMC
July 2020

tRNA-Derived Fragments Can Serve as Arginine Donors for Protein Arginylation.

Cell Chem Biol 2020 07 16;27(7):839-849.e4. Epub 2020 Jun 16.

Department of Biomedical Sciences, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address:

Arginyltransferase ATE1 mediates posttranslational arginylation and plays key roles in multiple physiological processes. ATE1 utilizes arginyl (Arg)-tRNA as the donor of Arg, putting this reaction into a direct competition with the protein synthesis machinery. Here, we address the question of ATE1- Arg-tRNA specificity as a potential mechanism enabling this competition in vivo. Using in vitro arginylation assays and Ate1 knockout models, we find that, in addition to full-length tRNA, ATE1 is also able to utilize short tRNA fragments that bear structural resemblance to tRNA-derived fragments (tRF), a recently discovered class of small regulatory non-coding RNAs with global emerging biological role. Ate1 knockout cells show a decrease in tRF generation and a significant increase in the ratio of tRNA:tRF compared with wild type, suggesting a functional link between tRF and arginylation. We propose that generation of physiologically important tRFs can serve as a switch between translation and protein arginylation.
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http://dx.doi.org/10.1016/j.chembiol.2020.05.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7409373PMC
July 2020

tRNA methylation: An unexpected link to bacterial resistance and persistence to antibiotics and beyond.

Wiley Interdiscip Rev RNA 2020 11 13;11(6):e1609. Epub 2020 Jun 13.

Department of Biochemistry & Molecular Biology, and Michael Smith Laboratories, University of British Columbia, Vancouver, Canada.

A major threat to public health is the resistance and persistence of Gram-negative bacteria to multiple drugs during antibiotic treatment. The resistance is due to the ability of these bacteria to block antibiotics from permeating into and accumulating inside the cell, while the persistence is due to the ability of these bacteria to enter into a nonreplicating state that shuts down major metabolic pathways but remains active in drug efflux. Resistance and persistence are permitted by the unique cell envelope structure of Gram-negative bacteria, which consists of both an outer and an inner membrane (OM and IM, respectively) that lay above and below the cell wall. Unexpectedly, recent work reveals that m G37 methylation of tRNA, at the N of guanosine at position 37 on the 3'-side of the tRNA anticodon, controls biosynthesis of both membranes and determines the integrity of cell envelope structure, thus providing a novel link to the development of bacterial resistance and persistence to antibiotics. The impact of m G37-tRNA methylation on Gram-negative bacteria can reach further, by determining the ability of these bacteria to exit from the persistence state when the antibiotic treatment is removed. These conceptual advances raise the possibility that successful targeting of m G37-tRNA methylation can provide new approaches for treating acute and chronic infections caused by Gram-negative bacteria. This article is categorized under: Translation > Translation Regulation RNA Processing > RNA Editing and Modification RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems.
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http://dx.doi.org/10.1002/wrna.1609DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7768609PMC
November 2020

Allele-specific RNA interference prevents neuropathy in Charcot-Marie-Tooth disease type 2D mouse models.

J Clin Invest 2019 12;129(12):5568-5583

The Jackson Laboratory, Bar Harbor, Maine, USA.

Gene therapy approaches are being deployed to treat recessive genetic disorders by restoring the expression of mutated genes. However, the feasibility of these approaches for dominantly inherited diseases - where treatment may require reduction in the expression of a toxic mutant protein resulting from a gain-of-function allele - is unclear. Here we show the efficacy of allele-specific RNAi as a potential therapy for Charcot-Marie-Tooth disease type 2D (CMT2D), caused by dominant mutations in glycyl-tRNA synthetase (GARS). A de novo mutation in GARS was identified in a patient with a severe peripheral neuropathy, and a mouse model precisely recreating the mutation was produced. These mice developed a neuropathy by 3-4 weeks of age, validating the pathogenicity of the mutation. RNAi sequences targeting mutant GARS mRNA, but not wild-type, were optimized and then packaged into AAV9 for in vivo delivery. This almost completely prevented the neuropathy in mice treated at birth. Delaying treatment until after disease onset showed modest benefit, though this effect decreased the longer treatment was delayed. These outcomes were reproduced in a second mouse model of CMT2D using a vector specifically targeting that allele. The effects were dose dependent, and persisted for at least 1 year. Our findings demonstrate the feasibility of AAV9-mediated allele-specific knockdown and provide proof of concept for gene therapy approaches for dominant neuromuscular diseases.
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http://dx.doi.org/10.1172/JCI130600DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6877339PMC
December 2019

How to Untie a Protein Knot.

Structure 2019 08;27(8):1190-1191

Department of Biochemistry and Molecular Biology, Thomas Jefferson University Philadelphia, PA 19107, USA. Electronic address:

The origin of protein backbone threading through a topological knot remains elusive. To understand the evolutionary origin of protein knots, in this issue of StructureKo et al. (2019) used circular permutation to untie a knotted protein. They showed that a domain-swapped dimer releases the knot and the associated high-energy state for substrate binding.
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http://dx.doi.org/10.1016/j.str.2019.07.008DOI Listing
August 2019

Loss-of-function mutations in Lysyl-tRNA synthetase cause various leukoencephalopathy phenotypes.

Neurol Genet 2019 Apr 18;5(2):e565. Epub 2019 Apr 18.

Department of Neurology (C.S., J.S., C.Z., J. Lu, J.X., S. Luo, J. Lin), Huashan Hospital, Fudan University, Shanghai, China; Baylor Genetic Laboratories (Y.J., Z.N., M.L.L., M.W., R.E.P., H.M., Y.Y.), Houston, TX; Department of Radiology (Y.L.), Huashan Hospital, Fudan University; Department of Pathology (Y.W., M.G.), Huashan Hospital, Fudan University, Shanghai, China; Department of Biochemistry and Molecular Pharmacology (M.L., K.D., Y.-M.H.), Thomas Jefferson University, Philadelphia, PA; Department of Human Genetics (S.N.O., A.A.), University of Michigan Medical School, Ann Arbor, MI; Department of Pediatrics and Department of Obstetrics and Gynecology (S.L.), University of Hawaii School of Medicine, Honolulu, HI; Department of Medical Oncology and Therapeutics Research (T.P.S.), Division of Clinical Cancer Genetics, City of Hope National Medical Center, Duarte, CA; Department of Molecular and Human Genetics (P.L.M., A.L.M., L.E., S.R.L., Z.N., M.L.L., J.A.R., M.W., R.E.P., H.M., J.A.R., Y.Y., V.W.Z.), Baylor College of Medicine, Houston, TX; and AmCare Genomics Lab (V.W.Z.), Guangzhou, China.

Objective: To expand the clinical spectrum of lysyl-tRNA synthetase () gene-related diseases, which so far includes Charcot-Marie-Tooth disease, congenital visual impairment and microcephaly, and nonsyndromic hearing impairment.

Methods: Whole-exome sequencing was performed on index patients from 4 unrelated families with leukoencephalopathy. Candidate pathogenic variants and their cosegregation were confirmed by Sanger sequencing. Effects of mutations on KARS protein function were examined by aminoacylation assays and yeast complementation assays.

Results: Common clinical features of the patients in this study included impaired cognitive ability, seizure, hypotonia, ataxia, and abnormal brain imaging, suggesting that the CNS involvement is the main clinical presentation. Six previously unreported and 1 known mutations were identified and cosegregated in these families. Two patients are compound heterozygous for missense mutations, 1 patient is homozygous for a missense mutation, and 1 patient harbored an insertion mutation and a missense mutation. Functional and structural analyses revealed that these mutations impair aminoacylation activity of lysyl-tRNA synthetase, indicating that defective KARS function is responsible for the phenotypes in these individuals.

Conclusions: Our results demonstrate that patients with loss-of-function mutations can manifest CNS disorders, thus broadening the phenotypic spectrum associated with KARS-related disease.
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http://dx.doi.org/10.1212/NXG.0000000000000316DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6515944PMC
April 2019

tRNA Methylation Is a Global Determinant of Bacterial Multi-drug Resistance.

Cell Syst 2019 04 10;8(4):302-314.e8. Epub 2019 Apr 10.

Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA 19107, USA. Electronic address:

Gram-negative bacteria are intrinsically resistant to drugs because of their double-membrane envelope structure that acts as a permeability barrier and as an anchor for efflux pumps. Antibiotics are blocked and expelled from cells and cannot reach high-enough intracellular concentrations to exert a therapeutic effect. Efforts to target one membrane protein at a time have been ineffective. Here, we show that mG37-tRNA methylation determines the synthesis of a multitude of membrane proteins via its control of translation at proline codons near the start of open reading frames. Decreases in mG37 levels in Escherichia coli and Salmonella impair membrane structure and sensitize these bacteria to multiple classes of antibiotics, rendering them incapable of developing resistance or persistence. Codon engineering of membrane-associated genes reduces their translational dependence on mG37 and confers resistance. These findings highlight the potential of tRNA methylation in codon-specific translation to control the development of multi-drug resistance in Gram-negative bacteria.
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http://dx.doi.org/10.1016/j.cels.2019.03.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6483872PMC
April 2019

Cysteinyl-tRNA Synthetase Mutations Cause a Multi-System, Recessive Disease That Includes Microcephaly, Developmental Delay, and Brittle Hair and Nails.

Am J Hum Genet 2019 03 26;104(3):520-529. Epub 2019 Feb 26.

Department of Clinical Genetics, Erasmus Medical Center, University Medical Center, 3015 GD Rotterdam, the Netherlands.

Aminoacyl-tRNA synthetases (ARSs) are essential enzymes responsible for charging tRNA molecules with cognate amino acids. Consistent with the essential function and ubiquitous expression of ARSs, mutations in 32 of the 37 ARS-encoding loci cause severe, early-onset recessive phenotypes. Previous genetic and functional data suggest a loss-of-function mechanism; however, our understanding of the allelic and locus heterogeneity of ARS-related disease is incomplete. Cysteinyl-tRNA synthetase (CARS) encodes the enzyme that charges tRNA with cysteine in the cytoplasm. To date, CARS variants have not been implicated in any human disease phenotype. Here, we report on four subjects from three families with complex syndromes that include microcephaly, developmental delay, and brittle hair and nails. Each affected person carries bi-allelic CARS variants: one individual is compound heterozygous for c.1138C>T (p.Gln380) and c.1022G>A (p.Arg341His), two related individuals are compound heterozygous for c.1076C>T (p.Ser359Leu) and c.1199T>A (p.Leu400Gln), and one individual is homozygous for c.2061dup (p.Ser688Glnfs2). Measurement of protein abundance, yeast complementation assays, and assessments of tRNA charging indicate that each CARS variant causes a loss-of-function effect. Compared to subjects with previously reported ARS-related diseases, individuals with bi-allelic CARS variants are unique in presenting with a brittle-hair-and-nail phenotype, which most likely reflects the high cysteine content in human keratins. In sum, our efforts implicate CARS variants in human inherited disease, expand the locus and clinical heterogeneity of ARS-related clinical phenotypes, and further support impaired tRNA charging as the primary mechanism of recessive ARS-related disease.
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http://dx.doi.org/10.1016/j.ajhg.2019.01.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6407526PMC
March 2019

Codon-Specific Translation by mG37 Methylation of tRNA.

Front Genet 2018 10;9:713. Epub 2019 Jan 10.

Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA, United States.

Although the genetic code is degenerate, synonymous codons for the same amino acid are not translated equally. Codon-specific translation is important for controlling gene expression and determining the proteome of a cell. At the molecular level, codon-specific translation is regulated by post-transcriptional epigenetic modifications of tRNA primarily at the wobble position 34 and at position 37 on the 3'-side of the anticodon. Modifications at these positions determine the quality of codon-anticodon pairing and the speed of translation on the ribosome. Different modifications operate in distinct mechanisms of codon-specific translation, generating a diversity of regulation that is previously unanticipated. Here we summarize recent work that demonstrates codon-specific translation mediated by the mG37 methylation of tRNA at CCC and CCU codons for proline, an amino acid that has unique features in translation.
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http://dx.doi.org/10.3389/fgene.2018.00713DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6335274PMC
January 2019

Author Correction: The selective tRNA aminoacylation mechanism based on a single G•U pair.

Nature 2018 12;564(7736):E37

RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, 230-0045, Japan.

In Fig. 1b of this Article, a U was inadvertently inserted after G15 in the D loop. The original Article has not been corrected.
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http://dx.doi.org/10.1038/s41586-018-0760-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6550308PMC
December 2018

tRNA 3'-amino-tailing for stable amino acid attachment.

RNA 2018 12 14;24(12):1878-1885. Epub 2018 Sep 14.

Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.

Amino acids are attached to the tRNA 3'-end as a prerequisite for entering the ribosome for protein synthesis. Amino acid attachment also gives tRNA access to nonribosomal cellular activities. However, the normal attachment is via an ester linkage between the carboxylic group of the amino acid and the 3'-hydroxyl of the terminal A76 ribose in tRNA. The instability of this ester linkage has severely hampered studies of aminoacyl-tRNAs. Although the use of 3'-amino-3'-deoxy A76 in a 3'-amino-tailed tRNA provides stable aminoacyl attachment via an amide linkage, there are multiple tailing protocols and the efficiency of each relative to the others is unknown. Here we compare five different tailing protocols in parallel, all dependent on the CCA-adding enzyme [CTP(ATP): tRNA nucleotidyl transferase; abbreviated as the CCA enzyme] to exchange the natural ribose with the modified one. We show that the most efficient protocol is achieved by the CCA-catalyzed pyrophosphorolysis removal of the natural A76 in equilibrium with the addition of the appropriate ATP analog to synthesize the modified 3'-end. This protocol for 3'-amino-tailing affords quantitative and stable attachment of a broad range of amino acids to tRNA, indicating its general utility for studies of aminoacyl-tRNAs in both canonical and noncanonical activities.
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http://dx.doi.org/10.1261/rna.068015.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6239183PMC
December 2018

Hypermorphic and hypomorphic AARS alleles in patients with CMT2N expand clinical and molecular heterogeneities.

Hum Mol Genet 2018 12;27(23):4036-4050

Department of Clinical Genetics, Leiden University Medical Center, Leiden, Netherlands.

Aminoacyl-tRNA synthetases (ARSs) are ubiquitously expressed enzymes implicated in several dominant and recessive disease phenotypes. The canonical function of ARSs is to couple an amino acid to a cognate transfer RNA (tRNA). We identified three novel disease-associated missense mutations in the alanyl-tRNA synthetase (AARS) gene in three families with dominant axonal Charcot-Marie-Tooth (CMT) disease. Two mutations (p.Arg326Trp and p.Glu337Lys) are located near a recurrent pathologic change in AARS, p.Arg329His. The third (p.Ser627Leu) is in the editing domain of the protein in which hitherto only mutations associated with recessive encephalopathies have been described. Yeast complementation assays demonstrated that two mutations (p.Ser627Leu and p.Arg326Trp) represent loss-of-function alleles, while the third (p.Glu337Lys) represents a hypermorphic allele. Further, aminoacylation assays confirmed that the third mutation (p.Glu337Lys) increases tRNA charging velocity. To test the effect of each mutation in the context of a vertebrate nervous system, we developed a zebrafish assay. Remarkably, all three mutations caused a pathological phenotype of neural abnormalities when expressed in zebrafish, while expression of the human wild-type messenger RNA (mRNA) did not. Our data indicate that not only functional null or hypomorphic alleles, but also hypermorphic AARS alleles can cause dominantly inherited axonal CMT disease.
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http://dx.doi.org/10.1093/hmg/ddy290DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6240730PMC
December 2018

Stabilization of Cyclin-Dependent Kinase 4 by Methionyl-tRNA Synthetase in p16-Negative Cancer.

ACS Pharmacol Transl Sci 2018 Sep 24;1(1):21-31. Epub 2018 Apr 24.

Medicinal Bioconvergence Research Center, Seoul National University, Suwon, 16229, Korea.

Although abnormal increases in the level or activity of cyclin-dependent kinase 4 (CDK4) occur frequently in cancer, the underlying mechanism is not fully understood. Here, we show that methionyl-tRNA synthetase (MRS) specifically stabilizes CDK4 by enhancing the formation of the complex between CDK4 and a chaperone protein. Knockdown of MRS reduced the CDK4 level, resulting in G0/G1 cell cycle arrest. The effects of MRS on CDK4 stability were more prominent in the tumor suppressor p16-negative cancer cells because of the competitive relationship of the two proteins for binding to CDK4. Suppression of MRS reduced cell transformation and the tumorigenic ability of a p16-negative breast cancer cell line . Further, the MRS levels showed a positive correlation with those of CDK4 and the downstream signals at high frequency in p16-negative human breast cancer tissues. This work revealed an unexpected functional connection between the two enzymes involving protein synthesis and the cell cycle.
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http://dx.doi.org/10.1021/acsptsci.8b00001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7089025PMC
September 2018

Selective terminal methylation of a tRNA wobble base.

Nucleic Acids Res 2018 04;46(7):e37

Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA 19107, USA.

Active tRNAs are extensively post-transcriptionally modified, particularly at the wobble position 34 and the position 37 on the 3'-side of the anticodon. The 5-carboxy-methoxy modification of U34 (cmo5U34) is present in Gram-negative tRNAs for six amino acids (Ala, Ser, Pro, Thr, Leu and Val), four of which (Ala, Ser, Pro and Thr) have a terminal methyl group to form 5-methoxy-carbonyl-methoxy-uridine (mcmo5U34) for higher reading-frame accuracy. The molecular basis for the selective terminal methylation is not understood. Many cmo5U34-tRNAs are essential for growth and cannot be substituted for mutational analysis. We show here that, with a novel genetic approach, we have created and isolated mutants of Escherichia coli tRNAPro and tRNAVal for analysis of the selective terminal methylation. We show that substitution of G35 in the anticodon of tRNAPro inactivates the terminal methylation, whereas introduction of G35 to tRNAVal confers it, indicating that G35 is a major determinant for the selectivity. We also show that, in tRNAPro, the terminal methylation at U34 is dependent on the primary m1G methylation at position 37 but not vice versa, indicating a hierarchical ranking of modifications between positions 34 and 37. We suggest that this hierarchy provides a mechanism to ensure top performance of a tRNA inside of cells.
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http://dx.doi.org/10.1093/nar/gky013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5909439PMC
April 2018

Transcription-translation coupling: direct interactions of RNA polymerase with ribosomes and ribosomal subunits.

Nucleic Acids Res 2017 Nov;45(19):11043-11055

Department of Biochemistry, University of California, Riverside, CA 92521, USA.

In prokaryotes, RNA polymerase and ribosomes can bind concurrently to the same RNA transcript, leading to the functional coupling of transcription and translation. The interactions between RNA polymerase and ribosomes are crucial for the coordination of transcription with translation. Here, we report that RNA polymerase directly binds ribosomes and isolated large and small ribosomal subunits. RNA polymerase and ribosomes form a one-to-one complex with a micromolar dissociation constant. The formation of the complex is modulated by the conformational and functional states of RNA polymerase and the ribosome. The binding interface on the large ribosomal subunit is buried by the small subunit during protein synthesis, whereas that on the small subunit remains solvent-accessible. The RNA polymerase binding site on the ribosome includes that of the isolated small ribosomal subunit. This direct interaction between RNA polymerase and ribosomes may contribute to the coupling of transcription to translation.
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http://dx.doi.org/10.1093/nar/gkx719DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5737488PMC
November 2017

Compound heterozygosity for loss-of-function GARS variants results in a multisystem developmental syndrome that includes severe growth retardation.

Hum Mutat 2017 10 14;38(10):1412-1420. Epub 2017 Jul 14.

Department of Human Genetics, University of Michigan, Ann Arbor, Michigan.

Aminoacyl-tRNA synthetases (ARSs) are ubiquitously expressed enzymes that ligate amino acids onto tRNA molecules. Genes encoding ARSs have been implicated in myriad dominant and recessive disease phenotypes. Glycyl-tRNA synthetase (GARS) is a bifunctional ARS that charges tRNA in the cytoplasm and mitochondria. GARS variants have been associated with dominant Charcot-Marie-Tooth disease but have not been convincingly implicated in recessive phenotypes. Here, we describe a patient from the NIH Undiagnosed Diseases Program with a multisystem, developmental phenotype. Whole-exome sequence analysis revealed that the patient is compound heterozygous for one frameshift (p.Glu83Ilefs*6) and one missense (p.Arg310Gln) GARS variant. Using in vitro and in vivo functional studies, we show that both GARS variants cause a loss-of-function effect: the frameshift variant results in depleted protein levels and the missense variant reduces GARS tRNA charging activity. In support of GARS variant pathogenicity, our patient shows striking phenotypic overlap with other patients having ARS-related recessive diseases, including features associated with variants in both cytoplasmic and mitochondrial ARSs; this observation is consistent with the essential function of GARS in both cellular locations. In summary, our clinical, genetic, and functional analyses expand the phenotypic spectrum associated with GARS variants.
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http://dx.doi.org/10.1002/humu.23287DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5599332PMC
October 2017

TrmD: A Methyl Transferase for tRNA Methylation With mG37.

Enzymes 2017;41:89-115. Epub 2017 Apr 12.

Center of New Technologies, University of Warsaw, Warsaw, Poland.

TrmD is an S-adenosyl methionine (AdoMet)-dependent methyl transferase that synthesizes the methylated mG37 in tRNA. TrmD is specific to and essential for bacterial growth, and it is fundamentally distinct from its eukaryotic and archaeal counterpart Trm5. TrmD is unusual by using a topological protein knot to bind AdoMet. Despite its restricted mobility, the TrmD knot has complex dynamics necessary to transmit the signal of AdoMet binding to promote tRNA binding and methyl transfer. Mutations in the TrmD knot block this intramolecular signaling and decrease the synthesis of mG37-tRNA, prompting ribosomes to +1-frameshifts and premature termination of protein synthesis. TrmD is unique among AdoMet-dependent methyl transferases in that it requires Mg in the catalytic mechanism. This Mg dependence is important for regulating Mg transport to Salmonella for survival of the pathogen in the host cell. The strict conservation of TrmD among bacterial species suggests that a better characterization of its enzymology and biology will have a broad impact on our understanding of bacterial pathogenesis.
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http://dx.doi.org/10.1016/bs.enz.2017.03.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6054489PMC
June 2019

Effect of Nascent Peptide Steric Bulk on Elongation Kinetics in the Ribosome Exit Tunnel.

J Mol Biol 2017 06 5;429(12):1873-1888. Epub 2017 May 5.

Department of Physiology, University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address:

All proteins are synthesized by the ribosome, a macromolecular complex that accomplishes the life-sustaining tasks of faithfully decoding mRNA and catalyzing peptide bond formation at the peptidyl transferase center (PTC). The ribosome has evolved an exit tunnel to host the elongating new peptide, protect it from proteolytic digestion, and guide its emergence. It is here that the nascent chain begins to fold. This folding process depends on the rate of translation at the PTC. We report here that besides PTC events, translation kinetics depend on steric constraints on nascent peptide side chains and that confined movements of cramped side chains within and through the tunnel fine-tune elongation rates.
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http://dx.doi.org/10.1016/j.jmb.2017.04.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5511029PMC
June 2017

A genetically encoded fluorescent tRNA is active in live-cell protein synthesis.

Nucleic Acids Res 2017 04;45(7):4081-4093

Department of Biochemistry and Molecular Biology, Thomas Jefferson University, 233 South 10th Street, Philadelphia, PA 19107, USA.

Transfer RNAs (tRNAs) perform essential tasks for all living cells. They are major components of the ribosomal machinery for protein synthesis and they also serve in non-ribosomal pathways for regulation and signaling metabolism. We describe the development of a genetically encoded fluorescent tRNA fusion with the potential for imaging in live Escherichia coli cells. This tRNA fusion carries a Spinach aptamer that becomes fluorescent upon binding of a cell-permeable and non-toxic fluorophore. We show that, despite having a structural framework significantly larger than any natural tRNA species, this fusion is a viable probe for monitoring tRNA stability in a cellular quality control mechanism that degrades structurally damaged tRNA. Importantly, this fusion is active in E. coli live-cell protein synthesis allowing peptidyl transfer at a rate sufficient to support cell growth, indicating that it is accommodated by translating ribosomes. Imaging analysis shows that this fusion and ribosomes are both excluded from the nucleoid, indicating that the fusion and ribosomes are in the cytosol together possibly engaged in protein synthesis. This fusion methodology has the potential for developing new tools for live-cell imaging of tRNA with the unique advantage of both stoichiometric labeling and broader application to all cells amenable to genetic engineering.
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http://dx.doi.org/10.1093/nar/gkw1229DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5397188PMC
April 2017

Initiator tRNA genes template the 3' CCA end at high frequencies in bacteria.

BMC Genomics 2016 12 8;17(1):1003. Epub 2016 Dec 8.

Department of Biochemistry and Molecular Biology, Thomas Jefferson University, 233 South 10th Street, BLSB 220, Philadelphia, PA, 19107, USA.

Background: While the CCA sequence at the mature 3' end of tRNAs is conserved and critical for translational function, a genetic template for this sequence is not always contained in tRNA genes. In eukaryotes and Archaea, the CCA ends of tRNAs are synthesized post-transcriptionally by CCA-adding enzymes. In Bacteria, tRNA genes template CCA sporadically.

Results: In order to understand the variation in how prokaryotic tRNA genes template CCA, we re-annotated tRNA genes in tRNAdb-CE database version 0.8. Among 132,129 prokaryotic tRNA genes, initiator tRNA genes template CCA at the highest average frequency (74.1%) over all functional classes except selenocysteine and pyrrolysine tRNA genes (88.1% and 100% respectively). Across bacterial phyla and a wide range of genome sizes, many lineages exist in which predominantly initiator tRNA genes template CCA. Convergent and parallel retention of CCA templating in initiator tRNA genes evolved in independent histories of reductive genome evolution in Bacteria. Also, in a majority of cyanobacterial and actinobacterial genera, predominantly initiator tRNA genes template CCA. We also found that a surprising fraction of archaeal tRNA genes template CCA.

Conclusions: We suggest that cotranscriptional synthesis of initiator tRNA CCA 3' ends can complement inefficient processing of initiator tRNA precursors, "bootstrap" rapid initiation of protein synthesis from a non-growing state, or contribute to an increase in cellular growth rates by reducing overheads of mass and energy to maintain nonfunctional tRNA precursor pools. More generally, CCA templating in structurally non-conforming tRNA genes can afford cells robustness and greater plasticity to respond rapidly to environmental changes and stimuli.
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http://dx.doi.org/10.1186/s12864-016-3314-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5143459PMC
December 2016
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