Scientific Report 2006

Molecular Biology

Development of the Genetic Code and Its Connection to Human Disease

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 is thought to have developed in the putative RNA world and thereby enabled the transition to the modern world of proteins. The early code was primitive and over many eons was refined. This refinement came from the acquisition of new activities by a group of proteins known as aminoacyl-tRNA synthetases. These proteins established the rules of the code through aminoacylation reactions, whereby each of the 20 amino acids is covalently joined to its cognate tRNA. The tRNA harbors the genetic code triplet associated with the specific amino acid that is joined to the tRNA.

Each amino acid has a single tRNA synthetase. The synthetases are thought to be among the earliest proteins, 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.

Detailed investigations of the structures and evolution of the aminoacyl-tRNA synthetases have provided a picture of the development of the genetic code and how the development was directed by the evolution of the synthetases and tRNAs. In previous research, we focused on the specifics of the molecular recognition of tRNAs and how the enzymes distinguish one tRNA from another to achieve accurate aminoacylation for a precise genetic code. During these studies, examination of a recent crystal structure of human tryptophanyl-tRNA synthetase (TrpRS) in complex with the tRNA for tryptophan (tRNA^Trp) revealed 2 states of the enzyme-tRNA complex (Fig. 1). In one state, the tRNA is entering the active site. In the other state, it has been charged (that is, tryptophan has been joined to tRNA^Trp in the aminoacylation reaction) and is dissociating from the enzyme.

Fig. 1. Crystal structures of uncharged tRNATrp associating with TrpRS (A) and charged tRNATrp dissociating from the enzyme (B). The location of the bound free amino acid (Trp) in the 2 active sites of the homodimer is indicated. The tRNA binds across both subunits.

During the long evolutionary development of aminoacyl-tRNA synthetases and their populating of every cell type, the enzymes adopted novel functions while keeping their canonical role as determinants 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 the canonical activities of the enzymes in translation, the 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 showed how the error-correction activity is essential for maintaining cell viability and how defects in this activity can lead to disease. In collaboration with S.L. Ackerman, Jackson Laboratories, Bar Harbor, Maine, we found that a single point mutation in mice leads to neurodegeneration (Fig. 2). In particular, Purkinje cells in the brain deteriorate and ataxia develops. This simple, heritable mutation is due to small amounts of misacylation of alanine-specific tRNA (tRNA^Ala) to generate, for example, serine attached to the tRNA. In this instance, small amounts of serine are incorporated in place of alanine in the polypeptides that are produced. Thus, a mild editing defect can lead to heritable neurologic disorders. A more severe defect in editing would doubtless be lethal and not sustained in the population.

Fig. 2. Pathologic changes in sticky mutant mice (A). B-D, Calbindin D-28 (Calb) immunohistochemistry of sagittal sections of cerebella from 3-week-old (B), 6-week-old (C), and 12-month-old (D) sti/sti mutant and 12-month-old wild-type (WT; E) mice. Cerebellar lobules are indicated by roman numerals. F-H, Hematoxylin and eosin staining of Purkinje cells (arrowheads) in lobule II of cerebella from 1-month-old (F) or 12-month-old (G) sti/sti mutant and 12-month-old wild-type (H) mice. I-N, Cleaved caspase 3 (Casp3) immunohistochemistry (I-K) and TUNEL analysis (L-N) of cerebella from 4-week-old mutant mice. Scale bars: For B-E, 500 μm; F-H, 50 μm; I-N, 10 μm.

In other collaborative studies with R. Burgess, Jackson Laboratories, we established a connection between glycyl-tRNA synthetase and Charcot-Marie-Tooth disease. A single point mutation in the synthetase leads to the disease. This mutation does not affect the aminoacylation activity of glycyl-tRNA synthetase. Instead, the results suggest that glycyl-tRNA synthetase has an additional function, possibly in neurologic development. Several examples of Charcot-Marie-Tooth disease due to mutations in glycyl-tRNA synthetase in humans have been found.

To better understand the molecular origins of this disease, we obtained crystals of human glycyl-tRNA synthetase that diffract to about 3 Å. A structure is being determined, and mutations found in humans will be mapped on the structure. This information will be used in conjunction with other assays and experiments to understand the connection between neurologic development, Charcot-Marie-Tooth disease, and glycyl-tRNA synthetase.

These results with glycyl-tRNA synthetase support the hypothesis that aminoacyl-tRNA synthetases in mammals are not only components of the translation apparatus but also a reservoir of cytokines with activities that are unmasked by specific activation events, such as alternative splicing or generation of specific fragments by proteolysis. Examples we are studying include tyrosyl- and tryptophanyl-tRNA synthetases. These are both procytokines that when split by alterative splicing or natural proteolysis, result in fragments that are active in signal transduction pathways. For example, a fragment of tryptophanyl-tRNA synthetase is a potent angiostatic agent.

Publications

Lee, J.W., Beebe, K., Nangle, L.A., Jang, J., Longo-Guess, C.M., Cook, S.A., Davisson, M.T., Sundberg, J.P., Schimmel, P., Ackerman, S.L. Editing-defective tRNA synthetase causes protein misfolding and neurodegeneration in the sticky mouse. Nature 443:50, 2006.

Nangle, L.A., Motta, C.M., Schimmel, P. Global effects of mistranslation from an editing defect in mammalian cells. Chem. Biol. 13:1091, 2006.

Reader, J.S., Ordoukhanian, P.T., Kim, J.-G., de Crécy-Lagard, V., Hwang, I.,
Farrand, S., Schimmel, P. Major biocontrol of plant tumors targets tRNA synthetase [published correction appears in Science 310:54, 2005]. Science 309:1533, 2005.

Schimmel, P., Beebe, K. From the RNA world to the theatre 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, 2005, p. 227.

Seburn, K.L., Nangle, L.A., Cox, G.A., Schimmel, P., Burgess, R.W. An active dominant mutant of glycyl-tRNA synthetase causes neuropathy in Charcot-Marie-Tooth 2D mouse model. Neuron 51:715, 2006.

Swairjo, M.A., Reddy, R.R., Lee, B., Van Lanen, S.G., Brown, S., de Crécy-Lagard, V., Iwata-Reuyl, D., Schimmel, P. Crystallization and preliminary x-ray characterization of the nitrile reductase QueF: a queosine-biosynthesis enzyme. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 61(Pt. 10):945, 2005.

Tamura, K., Schimmel, P.R. Chiral-selective aminoacylation of an RNA minihelix: mechanistic features and chiral suppression. Proc. Natl. Acad. Sci. U. S. A. 103:13750, 2006.

Tzima, E., Schimmel, P. Inhibition of tumor angiogenesis by a natural fragment of a tRNA synthetase. Trends Biochem. Sci. 31:7, 2006.

Waas, W.F., de Crécy-Lagard, V., Schimmel, P. Discovery of a gene family critical to wyosine base formation in a subset of phenylalanine-specific transfer RNAs. J. Biol. Chem. 280:37616, 2005.

Yang, X.-L., Otero, F.J., Ewalt, K.L., Liu, J., Swairjo, M.A., Kohrer, C., RajBhandary, U.L., Skene, R.J., McRee, D., Schimmel, P. Two conformations of a crystalline human tRNA synthetase-RNA complex: implications for protein synthesis. EMBO J. 25:2919, 2006.