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Ribonucleotide modifications perform a wide variety of roles in synthesis, turnover and functionality of tRNA molecules. The presence of particular chemical moieties can refine the internal interaction network within a tRNA molecule, influence its thermodynamic stability, contribute novel chemical properties and affect its decoding behavior during mRNA translation. As the lack of specific modifications in the anticodon stem and loop causes disrupted proteome homeostasis, diminished response to stress conditions, and the onset of human diseases, the underlying modification cascades have recently gained particular scientific and clinical interest. Nowadays, a complicated but conclusive image of the interconnectivity between different enzymatic modification cascades and their resulting tRNA modifications emerges. Here we summarize the current knowledge in the field, focusing on the known instances of cross talk among the enzymatic tRNA modification pathways and the consequences on the dynamic regulation of the tRNA modificome by various factors. This article is part of a Special Issue entitled: SI: Regulation of tRNA synthesis and modification in physiological conditions and disease edited by Dr. Boguta Magdalena.

The mechnisms of synthesis of mammalian selenocysteyl-tRNA were studied with murine active enzyme fractions. (75Se)Selenocysteyl-tRNA was synthesized in vitro by 500kDa protein from seryl-tRNA and (75Se)HSe-, which should be activated with ATP by 20kDa protein. This and other factors in mamalian systems are compared with those in Escherichia coli.

The mechnisms of synthesis of mammalian selenocysteyl-tRNA were studied with murine active enzyme fractions. (75Se)Selenocysteyl-tRNA was synthesized in vitro by 500kDa protein from seryl-tRNA and (75Se)HSe-, which should be activated with ATP by 20kDa protein. This and other factors in mamalian systems are compared with those in Escherichia coli.

Aminoacyl-tRNA (aa-tRNA) formation, an essential process in protein biosynthesis, is generally achieved by direct attachment of an amino acid to tRNA by the aa-tRNA synthetases. An exception is Gln-tRNA synthesis, which in eukaryotes is catalyzed by glutaminyl-tRNA synthetase (GlnRS), while most bacteria, archaea, and chloroplasts employ the transamidation pathway, in which a tRNA-dependent glutamate modification generates Gln-tRNA. Mitochondrial protein synthesis is carried out normally by mitochondrial enzymes and organelle-encoded tRNAs that are different from their cytoplasmic counterparts. Early work suggested that mitochondria use the transamidation pathway for Gln-tRNA formation. We found no biochemical support for this in Saccharomyces cerevisiae mitochondria, but demonstrated the presence of the cytoplasmic GlnRS in the organelle and its involvement in mitochondrial Gln-tRNA synthesis. In addition, we showed in vivo localization of cytoplasmic tRNAGln in mitochondria and demonstrated its role in mitochondrial translation. We furthermore reconstituted in vitro cytoplasmic tRNAGln import into mitochondria by a novel mechanism. This tRNA import mechanism expands our knowledge of RNA trafficking in the eukaryotic cell. These findings change our view of the evolution of organellar protein synthesis.

Transfer RNA is primarily synthesized by transcription by RNA polymerase and becomes the mature form via several steps: processing, splicing, CCA addition and posttranscriptional modification. The concentration of individual tRNA molecules is controlled in order to maintain cellular functions. Keywords: codon usage; gene-dosage effect; promoter; modified nucleotides; tRNA processing

Levels of transfer RNA (tRNA) were determined in liver, kidney, skeletal muscle, heart, and brain of young (35-day), adult (12-month) and old (24-month) female C57BL/6J mice. Kidney and liver showed little change in tRNA levels between young and adult mice, but the levels decreased in old mice. Skeletal muscle tRNA decreased steadily from young to old mice. Heart tRNA increased during maturation to adult organisms and then decreased in old individuals. Brain levels of tRNA increased steadily. No age-related change in the rate of transport of orotic acid into cells was observed. However, all tissues exhibited a decrease in uridine pools between adult and old mice. Most importantly, all tissues of aging mice showed a decrease in the rate of tRNA synthesis.

Publisher Summary This chapter discusses the synthesis, chemistry, and structural features of hypermodified nucleosides of tRNA. It deals with the direct effects of hypermodification altering nucleoside base-pairing and stacking properties as a result of inherent electronic and conformational changes. A brief inspection of the anticodon loop structure of tRNA Phe suggests the possibility of some indirect effects resulting from the interactions with the anticodon loop backbone, such as steric repulsion of bulk substituents, electrostatic repulsion of ionized groups (carboxyl and phosphodiester), hydrogen bonding, and Mg 2+ binding. Lack of accurate information about the tertiary structures of different tRNAs makes the evaluation of such indirect effects rather speculative. Some information can be gleaned from the simulation studies in which hypermodified nucleosides in their crystal structures are built into the yeast tRNA Phe anticodon loop at the positions of Gm 34 and of yW37, with the assumption that at least the main structural features are preserved in all tRNAs.

Rat liver methylating enzymes could methylate tRNA extracted from the livers of rats treated with 35 mg/100 g L-ethionine 19 h prior to sacrifice. 1-methylhypoxanthine and 3-methylcytosine were among the methylated bases synthesized in vitro . The synthesis of 3-methylcytosine was dependent on the presence of Mg++ although this ion inhibited the overall methylation of the tRNA.

The effect of spermine on the rates of ATP-pyrophosphate exchange and of formation of threonyl-tRNA by threonyl tRNA synthetase has been studied. It is shown that spermine alone has no effect on the overall reaction as well as on the rate of ATP pyrophosphate isotope exchange. At low Mg2+ concentration spermine activates 8-fold the ATP-pyrophosphate isotope exchange and 23-fold the threonyl tRNA synthesis. The possible role of spermine in this activation is discussed.