Scientific Report 2005

Molecular Biology

Components of the Genetic Code in Translation, Cell Biology, and Medicine

P. Schimmel, J. Bacher, K. Beebe, Z. Druzina, K. Ewalt, M. Kapoor, E. Merriman, C. Motta, L. Nangle, F. Otero, J. Reader, R. Reddy, M. Swairjo, K. Tamura, E. Tzima, W. Waas, X.-L. Yang

The genetic code was established in the transition from the RNA world to the theater of proteins. The code is an algorithm, matching each amino acid with a nucleotide triplet. The matching of triplets with amino acids occurs through aminoacylation reactions in which enzymes known as aminoacyl-tRNA synthetases catalyze attachment of each amino acid to its cognate tRNA. Each tRNA, in turn, has an anticodon nucleotide triplet that defines the amino acid–nucleotide triplet relationship of the code.

Each amino acid has a single tRNA synthetase. The synthetases are thought to be among the earliest proteins and, as such, essential components of the translation apparatus that established the genetic code and that were present in the last common ancestor of the universal tree of life. As the tree developed and branched into the 3 great kingdoms—archaebacteria, bacteria, and eukaryotes—the enzymes were incorporated into every cell type of every organism. During this long evolutionary period and populating of every cell, the enzymes adopted novel functions while keeping their canonical role as determinates of the genetic code. Related to their central role, the enzymes acquired novel domains enabling them to correct errors of aminoacylation and thereby ensure the stringent accuracy of the code. Unrelated to their canonical activity in translation, their expanded functions include regulation of transcription and translation in bacteria, RNA splicing in fungal organisms, and cytokine signaling in mammalian cells. These novel functions connect translation to other central pathways that control growth, development, and regulation of all cell types.

Recently, we have focused on 2 of the expanded functions that have connections to disease and medicine. One function is the editing activity of the synthetases. Mutations in the editing domain of a specific tRNA synthetase cause ambiguity in the genetic code and result in subtle missense substitutions in proteins throughout the organism (Fig. 1).

Fig. 1. Aminoacyl-tRNA synthetases catalyze the attachment of a noncognate amino acid onto tRNA. A distinct hydrolytic second site prevents these substrates from being released for use in protein synthesis. Mutations within the editing site result in the inability to clear noncognate amino acids from the tRNA. These errors in proofreading ultimately lead to incorporation of wrong amino acids into a growing polypeptide. The final result of accumulation of proteins with errors in their primary sequences is cell death.

These changes, in turn, cause global changes in protein function. Such changes can, in principle, lead to specific diseases, such as autoimmune disorders. Indeed, specific changes in the phenotypes of mammalian cells in culture occur when an editing-defective synthetase is present.

In mammalian cells, tyrosyl- and tryptophanyl-tRNA synthetases are procytokines. When these synthetases are split by alterative splicing or natural proteolysis, specific fragments are released. These fragments are active in signal transduction pathways. For example, T2-TrpRS, a fragment of tryptophanyl-tRNA synthetase, is a potent angiostatic agent. In collaborative experiments with M. Friedlander, Department of Cell Biology, we found that T2-TrpRS arrested angiogenesis in the retina in neonatal mice. The fragment is so effective in arresting angiogenesis that it is now being introduced into a clinical setting for the treatment of blindness caused by macular degeneration. In other research, we are focusing on the usefulness of T2-TrpRS for treatment of highly vascularized tumors.

To understand the antiangiogenic activity of T2-TrpRS, we are identifying the cell signaling pathway involved. Recent experiments indicated that vascular endothelial cell cadherin (VE-cadherin), a calcium-dependent adhesion molecule specifically expressed in endothelial cells and essential for normal vascular development, binds directly to T2-TrpRS. This binding, in turn, blocks the proangiogenic activity of vascular endothelial cell growth factor (Fig. 2).

Fig. 2. Schematic illustration of proposed model for how T2-TrpRS (T2) interacts with VE-cadherin and blocks signaling pathways for vascular endothelial cell growth vector (VEGF) and its receptor (VEGFR2).

Currently, we are examining the mechanism of signaling by T2-TrpRS after it is bound to VE-cadherin and the mechanism of export of T2-TrpRS from the cytoplasm to the cell surface. In addition, on the basis of x-ray structures, we proposed a structure-based mechanism for cytokine activation: the structural changes that occur when tryptophanyl- and tyrosyl-tRNA synthetases are split into specific fragments that convert the synthetases to cytokines.

In other research, we are investigating the critical steps in the transition from the RNA world to the theater of proteins. Recent findings established a plausible scenario for the selection of L- rather than D-amino acids as the building blocks for proteins in all life forms. Using amino acids activated in a way similar to the way in which modern amino acids are activated, we showed chiral-selective aminoacylation of tRNA-like molecules. We are using x-ray analysis to understand the structural basis of the chiral selectivity.

Publications

Bacher, J.M., de Crécy-Lagard, V., Schimmel, P. Inhibited cell growth and protein functional changes from an editing-defective tRNA synthetase. Proc. Natl. Acad. Sci. U. S. A. 102:1697, 2005.

Ewalt, K.L., Schimmel, P. Protein biosynthesis: tRNA synthetases. In: Encyclopedia of Biological Chemistry. Lennarz, W.J., Lane, M.D. (Eds.). Academic Press, San Diego, 2004, p. 263.

Ewalt, K.L., Yang, X.-L., Otero, F.J., Liu, J., Slike, B., Schimmel, P. Variant of human enzyme sequesters reactive intermediate. Biochemistry 44:4216, 2005.

Metzgar, D., Bacher, J.M., Pezo, V., Reader, J., Doring, V., Schimmel, P., Marlière, P., de Crécy-Lagard, V. Acinetobacter sp ADP1: an ideal model organism for genetic analysis and genome engineering. Nucleic Acid Res. 32:5780, 2004.

Nordin, B.E., Schimmel, P. Isoleucyl-tRNA synthetases. In: Aminoacyl-tRNA Synthetases. Ibba, M., Francklyn, C., Cusack, S. (Eds.). Landes Bioscience/Eurekah.com, Georgetown, TX, 2005, p. 24.

Ribas de Pouplana, L., Musier-Forsyth, K., Schimmel, P. Alanyl-tRNA synthetases. In: Aminoacyl-tRNA Synthetases. Ibba, M., Francklyn, C., Cusack, S. (Eds.). Landes Bioscience/Eurekah.com, Georgetown, TX, 2005, p. 241.

Ribas de Pouplana, L., Schimmel, P. Aminoacylations of tRNAs: record-keepers for the genetic code. In: Protein Synthesis and Ribosome Structure: Translating the Genome. Nierhaus, K.H., Wilson, D.N. (Eds.), Wiley-VCH, New York, 2004, p. 169.

Schimmel, P. Genetic code. In: McGraw-Hill Encyclopedia of Science and Technology, 10th ed. McGraw-Hill, New York, in press.

Schimmel, P., Beebe, K. From the RNA world to the theater of proteins. In: The RNA World, 3rd ed. Gesteland, R.R., Cech, T.R., Atkins, J.F. (Eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, in press.

Schimmel, P., Ewalt, K. Translation silenced by fused pair of tRNA synthetases. Cell 119:147, 2004.

Schimmel, P., Söll, D. The world of aminoacyl-tRNA synthetases. In: Aminoacyl-tRNA Synthetases. Ibba, M., Francklyn, C., Cusack, S. (Eds.). Landes Bioscience/Eurekah.com, Georgetown, TX, 2005, p. 1.

Swairjo, M.A., Schimmel, P. Breaking sieve for steric exclusion of a noncognate amino acid from active site of a tRNA synthetase. Proc. Natl. Acad. Sci. U. S. A. 102:988, 2005.

Tamura, K., Schimmel, P. Non-enzymatic aminoacylation of an RNA minihelix with an aminoacyl phosphate oligonucleotide. Nucleic Acids Symp. Ser. 48:269, 2004.

Tang, H.-L., Yeh, L.-S., Chen, N.-K., Ripmaster, T.L., Schimmel, P., Wang, C.-C. Translation of a yeast mitochondrial tRNA synthetase initiated at redundant non-AUG codons. J. Biol. Chem. 279:49656, 2004.

Tzima, E., Reader, J.S., Irani-Tehrani, M., Ewalt, K.L., Schwartz, M.A., Schimmel, P. VE-cadherin links tRNA synthetase cytokine to anti-angiogenic function. J. Biol. Chem. 280:2405, 2005.