Effects of modifying the tRNA(3Lys) anticodon on the initiation of human immunodeficiency virus type 1 reverse transcription

tRNA(3Lys) is a primer for reverse transcription in human immunodeficiency virus type 1 (HIV-1), and the anticodon of tRNA(3Lys) has been implicated in playing a role in both its placement onto the HIV-1 genome and its interaction with HIV-1 reverse transcriptase (RT). In this work, the anticodon in a tRNA(3Lys) gene was changed from UUU to CUA (tRNA(3Lys)Su+) or, in addition, G-73 was altered to A (tRNA(3Lys)Su+G73A). COS-7 cells were transfected with either wild-type or mutant tRNA(3Lys) genes, and both the wild-type and mutant tRNA(3Lys) produced were purified by using immobilized tRNA-specific hybridization probes. Each mutant tRNA(3Lys) was tested for its ability to prime reverse transcription in vitro, either alone or in competition with wild-type tRNA(3Lys). Short RT extensions of wild-type and mutant tRNALys could be distinguished from each other by their different mobilities in one-dimensional single-stranded conformation polymorphism polyacrylamide gel electrophoresis. These reverse transcription products show that heat-annealed tRNA(3Lys)Su+ has the same ability as heat-annealed wild-type tRNA(3Lys) to prime RT and competes equally well with wild-type tRNA(3Lys) for priming RT. tRNA(3Lys)Su+G73A has 60% of the wild-type ability to prime RT but competes poorly with wild-type tRNA(3Lys) for priming RT. However, the priming abilities of wild-type and mutant tRNA(3) are quite different when in vivo-placed tRNA is examined. HIV-1 produced in COS cells transfected with a plasmid containing both the HIV-1 proviral DNA and DNA coding for tRNA(3Lys)Su+ contains both endogenous, cellular wild-type tRNA(3Lys) and mutant tRNA(3Lys). When total viral RNA is used as the source of primer tRNA placed onto the genomic RNA in vivo, only wild-type tRNA(3Lys) is used as a primer. If the total viral RNA is first heated and exposed to hybridizing conditions, then both the wild-type and mutant tRNA(3Lys) act as primers for RT. These results indicate that the tRNA(3Lys)Su+ packaged into the virions is unable to act as a primer for RT, and a model is proposed to explain the disparate results between heat-annealed and in vivo-placed primer tRNA.

Estudo do papel da proteína eIF5A na elongação da tradução em S. cerevisiae: análise da relação funcional com o trna de alanina e sua participação na síntese proteica geral da célula

Pós-graduação em Biotecnologia - IQ

Mutations in the Mitochondrial Methionyl-tRNA Synthetase Cause a Neurodegenerative Phenotype in Flies and a Recessive Ataxia (ARSAL) in Humans

An increasing number of genes required for mitochondrial biogenesis, dynamics, or function have been found to be mutated in metabolic disorders and neurological diseases such as Leigh Syndrome. In a forward genetic screen to identify genes required for neuronal function and survival in Drosophila photoreceptor neurons, we have identified mutations in the mitochondrial methionyl-tRNA synthetase, Aats-met, the homologue of human MARS2. The fly mutants exhibit age-dependent degeneration of photoreceptors, shortened lifespan, and reduced cell proliferation in epithelial tissues. We further observed that these mutants display defects in oxidative phosphorylation, increased Reactive Oxygen Species (ROS), and an upregulated mitochondrial Unfolded Protein Response. With the aid of this knowledge, we identified MARS2 to be mutated in Autosomal Recessive Spastic Ataxia with Leukoencephalopathy (ARSAL) patients. We uncovered complex rearrangements in the MARS2 gene in all ARSAL patients. Analysis of patient cells revealed decreased levels of MARS2 protein and a reduced rate of mitochondrial protein synthesis. Patient cells also exhibited reduced Complex I activity, increased ROS, and a slower cell proliferation rate, similar to Drosophila Aats-met mutants.

Enzyme tRNA interaction and (t)RNA conformation probed via environmentally sensitive cyanine dyes

Nukleosidmodifikationen beeinflussen Dynamik und Konformation von RNArnund sind epigenetisch wirksam. Wenig verstanden sind konformationelle Dynamik und enzymatische Erkennung von tRNA, sowie der Einfluss des mutmaßlichen kovalenten Inhibitors 5-Fluorouridine (5FU) auf Y Synthasen, die Pseudouridin (Y) erzeugen. Frühere Arbeiten nutzten mit den Fluorophoren Cy3 und Cy5rnmarkierte tRNA, um diese Fragen zu adressieren.rnDie vorliegende Arbeit weitet Cy3-Cy5-Markierung auf Hefe tRNArnPhernaus undrnnutzt Thermophorese und fortschrittliche Fluoreszenzspektroskopie. In der Thermophorese zeigte sich eine hohe Toleranz gegenüber Fluoreszenzmarkierung beirngleichzeitiger Erhöhung der Cy5 Fluoreszenz durch Enzymbindung. Zudem konnte die Konformation verschiedener Mutanten human mitochondrialer tRNArnLysrnund die Bindung von SAM durch SAM-I Riboswitch RNA untersucht werden.rnUm etwaige Unterschiede in der Interaktion von Y55 Synthase TruB mit Cy5-gelabelter U55- bzw. 5FU55-tRNA aufzudecken, wurde eine Kombination ausrnThermophorese, zeit- und polarisationsaufgelöster Fluoreszenzspektroskopie undrn’gel shift’ Experimenten genutzt. Alle Ergebnisse zeigten übereinstimmend einernreversible Bindung ähnlicher Affinität für beide tRNAs und widersprechen somit einer kovalenten Inhibition durch 5FU. Folgerichtig wurde der SDS-stabilernKomplex von TruB mit 5FU-tRNA neu evaluiert, da er bisher als kovalent interpretiert wurde. Es erfolgte eine schnelle Komplexbildung in hoher Ausbeute auchrnfür schlechte Substrate, außerdem ließ sich die Komplexausbeute nicht durch andere Reaktionsbedingungen beeinflussen. Somit kann der SDS stabile Komplexrnnur den ersten, nicht-kovalenten Kontakt von Enzym und 5FU55-tRNA darstellen und repräsentiert kein kovalentes Addukt späterer Katalyse.

Investigations on DNA methylation by Dnmt2 and impact of tRNA modifications on TLR7 stimulation

The incorporation of modified nucleotides into ribonucleic acids (RNAs) is important for their structure and proper function. These modifications are inserted by distinct catalytic macromolecules one of them being Dnmt2. It methylates the Cytidine (C) at position 38 in tRNA to 5-methylcytidine (m5C). Dnmt2 has been a paradigm in this respect, because all of its nearest neighbors in evolution are DNA-cytosine C5-methyltransferases and methylate DNA, while its (own) DNA methyltransferase activity is the subject of controversial reports with rates varying between zero and very weak. This work determines whether the biochemical potential for DNA methylation is present in the enzyme. It was discovered that DNA fragments, when presented as covalent RNA:DNA hybrids in the structural context of a tRNA, can be more efficiently methylated than the corresponding natural tRNA substrate. Additional minor deviations from a native tRNA structure that were seen to be tolerated by Dnmt2 were used for a stepwise development of a composite system of guide RNAs that enable the enzyme to perform cytidine methylation on single stranded DNA in vitro. Furthermore, a proof-of-principle is presented for utilizing the S-adenosyl methionine-analog cofactor SeAdoYn with Dnmt2 to search for new possible substrates in a SELEX-like approach.rnIn innate immunity, nucleic acids can function as pathogen associated molecular patterns (PAMPs) recognized by pattern recognition receptors (PRRs). The modification pattern of RNA is the discriminating factor for toll-like receptor 7 (TLR7) to distinguish between self and non-self RNA of invading pathogens. It was found that a 2'-O-methylated guanosine (Gm) at position18, naturally occurring at this position in some tRNAs, antagonizes recognition by TLR7. In the second part of this work it is pointed out, that recognition extends to the next downstream nucleotide and the effectively recognized molecular detail is actually a methylated dinucleotide. The immune silencing effect of the ribose methylation is most pronounced if the dinucleotide motif is composed of purin nucleobases whereas pyrimidines diminish the effect. Similar results were obtained when the Gm modification was transposed into other tRNA domains. Point mutations abolishing base pairings important for a proper tertiary structure had no effect on the immune stimulatory potential of a Gm modified tRNA. Taken together these results suggest a processive type of RNA inspection by TLR7.rn

Mitochondrial tRNA(Leu(UUR)) mutation m.3302A > G presenting as childhood-onset severe myopathy: threshold determination through segregation study

Mitochondrial tRNA(Leu(UUR)) mutation m.3302A > G is associated with respiratory chain complex I deficiency and has been described as a rare cause of mostly adult-onset slowly progressive myopathy. Five families with 11 patients have been described so far; 5 of them died young due to cardiorespiratory failure. Here, we report on a segregation study in a family with an index patient who already presented at the age of 18 months with proximal muscular hypotonia, abnormal fatigability, and lactic acidosis. This early-onset myopathy was rapidly progressive. At 8 years, the patient is wheel-chair bound, requires nocturnal assisted ventilation, and suffers from recurrent respiratory infections. Severe complex I deficiency and nearly homoplasmy for m.3302A > G were found in muscle. We collected blood, hair, buccal swabs and muscle biopsies from asymptomatic adults in this pedigree and determined heteroplasmy levels in these tissues as well as OXPHOS activities in muscle. All participating asymptomatic adults had normal OXPHOS activities. In contrast to earlier reports, we found surprisingly little variation of heteroplasmy levels in different tissues of the same individual. Up to 45% mutation load in muscle and up to 38% mutation load in other tissues were found in non-affected adults. The phenotypic spectrum of tRNA(Leu(UUR)) m.3302A > G mutation seems to be wider than previously described. A threshold of more than 45% heteroplasmy in muscle seems to be necessary to alter complex I activity leading to clinical manifestation. The presented data may be helpful for prognostic considerations and counseling in affected families.

Mitochondrial tRNA import and its consequences for mitochondrial translation

The mitochondrial genomes of most eukaryotes lack a variable number of tRNA genes. This lack is compensated for by import of a small fraction of the corresponding cytosolic tRNAs. There are two broad mechanisms for the import of tRNAs into mitochondria. In the first one, the tRNA is coimported together with a mitochondrial precursor protein along the protein import pathway. It applies to the yeast tRNA(Lys) and has been elucidated in great detail. In the second more vaguely defined mechanism, which is mainly found in plants and protozoa, tRNAs are directly imported independent of cytosolic factors. However, results in plants indicate that direct import of tRNAs may nevertheless require some components of the protein import machinery. All imported tRNAs in all systems are of the eukaryotic type but need to be functionally integrated into the mitochondrial translation system of bacterial descent. For some tRNAs, this is not trivial and requires unique evolutionary adaptations.

Point mutation tRNA(Ser(UCN)) in a child with hearing loss and myoclonus epilepsy

We report on a family with a 12-year-old boy who suffered from a maternally inherited syndrome characterized by a combination of sensorineural hearing loss, myoclonus epilepsy, ataxia, severe psychomotor retardation, short stature, and diabetes mellitus. First, he showed a muscular hypotonia with hearing loss; later, he developed a myoclonus epilepsy, growth failure, and severe psychomotor retardation. At the age of 10 years, he developed diabetes mellitus. After initiation of combined ubiquinone and vitamin C treatment, we observed a progression in psychomotor development. Lactate and pyruvate levels in blood and cerebrospinal fluid were normal. No ragged red fibers or ultrastructural abnormalities were seen in a skeletal muscle biopsy. Biochemical assays of respiratory chain complex activities revealed decreased activity of complexes I and IV. By sequence analysis of mitochondrial DNA encoding transfer ribonucleic acids (RNAs), a homoplasmic T to C substitution at nucleotide position 7512 was found affecting a highly conserved base pair in the tRNA(ser(UCN)) acceptor stem. Asymptomatic family members of the maternal line were heteroplasmic for the mutation in blood samples. Analysis of mitochondrial DNA in patients with hearing loss and myoclonus epilepsy is recommended, even in the absence of laboratory findings. Therapeutically, ubiquinone and antioxidants can be beneficial.

Handling mammalian mitochondrial tRNAs and aminoacyl-tRNA synthetases for functional and structural characterization

The mammalian mitochondrial (mt) genome codes for only 13 proteins, which are essential components in the process of oxidative phosphorylation of ADP into ATP. Synthesis of these proteins relies on a proper mt translation machinery. While 22 tRNAs and 2 rRNAs are also coded by the mt genome, all other factors including the set of aminoacyl-tRNA synthetases (aaRSs) are encoded in the nucleus and imported. Investigation of mammalian mt aminoacylation systems (and mt translation in general) gains more and more interest not only in regard of evolutionary considerations but also with respect to the growing number of diseases linked to mutations in the genes of either mt-tRNAs, synthetases or other factors. Here we report on methodological approaches for biochemical, functional, and structural characterization of human/mammalian mt-tRNAs and aaRSs. Procedures for preparation of native and in vitro transcribed tRNAs are accompanied by recommendations for specific handling of tRNAs incline to structural instability and chemical fragility. Large-scale preparation of mg amounts of highly soluble recombinant synthetases is a prerequisite for structural investigations that requires particular optimizations. Successful examples leading to crystallization of four mt-aaRSs and high-resolution structures are recalled and limitations discussed. Finally, the need for and the state-of-the-art in setting up an in vitro mt translation system are emphasized. Biochemical characterization of a subset of mammalian aminoacylation systems has already revealed a number of unprecedented peculiarities of interest for the study of evolution and forensic research. Further efforts in this field will certainly be rewarded by many exciting discoveries.

Aminoacyl-tRNA synthetases: Probing the molecular basis of catalysis and discrimination

Aminoacyl-tRNA synthetases (RSs) are responsible for the essential connection of amino acids with trinucleotide sequences of tRNA's. The RS family constitutes two structurally dissimilar groups of proteins, class I and class II. Methionyl-tRNA synthetase (MetRS) and isoleucyl-tRNA synthetase (IleRS), both members of class I, were the focus of this work. Both enzymes are zinc-containing proteins; show a high degree of amino acid specificity; and edit activated noncognate amino acids, thereby ensuring the fidelity of the genetic code. The goals of this work were to further delineate the molecular basis of catalysis and discrimination in these enzymes by mapping active site geometries using high-resolution nuclear magnetic resonance spectroscopy (NMR).^ Internuclear distances obtained from transferred nuclear Overhauser effects were used to define the conformations of Mg($\alpha$,$\beta$-methylene)ATP bound to E. coli MetRS and E. coli IleRS in multiple complexes. Identical conformations were found for the bound ATP. Thus, the predicted structural homology between IleRS and MetRS is supported by consensus enzyme-bound nucleotide conformations. The conformation of the bound nucleotide is not sensitive to occupation of the amino acid site of MetRS or IleRS. Therefore, conformational changes known to occur in the synthetases upon ligand binding appear not to alter the bound conformation of the adenosine portion of the nucleotide. Nuclear Overhauser effects on the substrate amino acid L-selenomethionine were also used to evaluate the enzyme-bound conformation of the cognate amino acid. The amino acid assumes a conformation which is consistent with a proposed editing mechanism.^ The E. coli MetRS was shown to catalyze amino acid $\alpha$-proton exchange in the presence of deuterium oxide of all cognate amino acids. It is proposed that the enzyme-bound zinc coordinates the $\alpha$-carboxylate of the amino acid, rendering the $\alpha$-proton more acidic. An enzymic base is responsible for exchange of the $\alpha$-proton. This proposal suggests that the enzyme-bound zinc may have a role in amino acid discrimination in MetRS. However, the role of this exchange reaction in catalysis remains unknown. ^