Alexey D. Wolfson

Diadenosine oligophosphates (ApnA), a novel class of signalling molecules?

The diadenosine oligophosphates (Ap n A) were discovered in the mid‐sixties in the course of studies on aminoacyl‐tRNA synthetases (aaRS). Now, more than 30 years later, about 300 papers have been published around these substances in attempt to decipher their role in cells. Recently, Ap n A have emerged as intracellular and extracellular signalling molecules implicated in the maintenance and regulation of vital cellular functions and become considered as second messengers. Great variety of physiological and pathological effects in mammalian cells was found to be associated with alterations of Ap n A levels (n from 2 to 6) and Ap3A/Ap4A ratio. Cell differentiation and apoptosis have substantial and opposite effects on Ap3A/Ap4A ratio in cultured cells. A human Ap3A hydrolase, Fhit, appeared to be involved in protection of cells against tumourigenesis. Ap3A is synthesised by mammalian u synthetase (TrpRS) which in contrast to most other aaRS is unable to synthesise Ap4A and is an interferon‐inducible protein. Moreover, Ap3A appeared to be a preferred substrate for 2‐5A synthetase, also interferon‐inducible, priming the synthesis of 2′ adenylated derivatives of Ap3A, which in turn may serve as substrates of Fhit. Tumour suppressor activity of Fhit is assumed to be associated with involvement of the Fhit·Ap3A complex in cytokine signalling pathway(s) controlling cell proliferation. The Ap n A family is potentially a novel class of signal‐transducing molecules whose functions are yet to be determined.

Modulation of tRNAAla identity by inorganic pyrophosphatase

A highly sensitive assay of tRNA aminoacylation was developed that directly measures the fraction of aminoacylated tRNA by following amino acid attachment to the 3′-32P-labeled tRNA. When applied to Escherichia coli alanyl-tRNA synthetase, the assay allowed accurate measurement of aminoacylation of the most deleterious mutants of tRNAAla. The effect of tRNAAla identity mutations on both aminoacylation efficiency (kcat/KM) and steady-state level of aminoacyl-tRNA was evaluated in the absence and presence of inorganic pyrophosphatase and elongation factor Tu. Significant levels of aminoacylation were achieved for tRNA mutants even when the kcat/KM value is reduced by as much as several thousandfold. These results partially reconcile the discrepancy between in vivo and in vitro analysis of tRNAAla identity.

Arginyltransferase Is an ATP-Independent Self-Regulating Enzyme that Forms Distinct Functional Complexes In Vivo

Posttranslational arginylation mediated by arginyl transferase (ATE1) plays an important role in cardiovascular development, cell motility, and regulation of cytoskeleton and metabolic enzymes. This protein modification was discovered decades ago, however, the arginylation reaction and the functioning of ATE1 remained poorly understood because of the lack of good biochemical models. Here, we report the development of an in vitro arginylation system, in which ATE1 function and molecular requirements can be tested using purified recombinant ATE1 isoforms supplemented with a controlled number of components. Our results show that arginylation reaction is a self-sufficient, ATP-independent process that can affect different sites in a polypeptide and that arginyl transferases form different molecular complexes in vivo, associate with components of the translation machinery, and have distinct, partially overlapping subsets of substrates, suggesting that these enzymes play different physiological functions.

Stable tRNA-based phylogenies using only 76 nucleotides

tRNAs are among the most ancient, highly conserved sequences on earth, but are often thought to be poor phylogenetic markers because they are short, often subject to horizontal gene transfer, and easily change specificity. Here we use an algorithm now commonly used in microbial ecology, UniFrac, to cluster 175 genomes spanning all three domains of life based on the phylogenetic relationships among their complete tRNA pools. We find that the overall pattern of similarities and differences in the tRNA pools recaptures universal phylogeny to a remarkable extent, and that the resulting tree is similar to the distribution of bootstrapped rRNA trees from the same genomes. In contrast, the trees derived from tRNAs of identical specificity or of individual isoacceptors generally produced trees of lower quality. However, some tRNA isoacceptors were very good predictors of the overall pattern of organismal evolution. These results show that UniFrac can extract meaningful biological patterns from even phylogenies with high level of statistical inaccuracy and horizontal gene transfer, and that, overall, the pattern of tRNA evolution tracks universal phylogeny and provides a background against which we can test hypotheses about the evolution of individual isoacceptors.

New competition in RNA regulation

An additional layer of RNA regulation in which RNAs encoded by genes and pseudogenes compete for microRNAs could offer new opportunities for oligonucleotide therapeutics.

Occurrence of the aminoacyl-tRNA synthetases in high-molecular weight complexes correlates with the size of substrate amino acids

One of the distinctive and mysterious features of mammalian aminoacyl‐tRNA synthetases (AARSs) is the existence of stable high‐molecular weight complexes containing 10 out of 20 AARSs. The composition and structure of these complexes are conserved among multicellular animals. No specific function associated with these structures has been found, and there is no evident rationale for a particular separation of AARSs in “complex‐bound” and “free” forms. We have demonstrated a strong association between the occurrence of AARSs in the complexes and the volume of their substrate amino acids. The significance of this association is discussed in terms of the structural organization of translation in the living cell.

Crucial role of pyrophosphate in the aminoacylation of E. coli tRNAPhe by yeast phenylalanyl-tRNA synthetase

Rapid inactivation of the yeast phenylalanyl‐tRNA synthetase in the course of aminoacylation of the heterologous E. coli tRNAPhe is observed. This inactivation occurs due to the formation of the tight complex of the enzyme with the pyrophosphate formed during the aminoacylation reaction. This complex is shown to be the normal intermediate of the reaction. Possible inactivation mechanism and correlation between structural differences of yeast and E. coli tRNAsPhe with the changes in the enzymatic mechanism of aminoacylation are discussed.