Little is known about the role of specific base modifications of transfer RNAs. Wyosine bases are tRNA(Phe)-specific modifications that are distinguished by differentiated, lateral side chains and base methylations appended to the core ring structure of a universally conserved G37, adjacent to the anticodon of Phe tRNAs. Based on previous data, we hypothesized that this modification was needed for -1 frameshifting. Using a reporter system incorporating a SCV-LA yeast virus slippery site for detecting -1 frameshifts in vivo, yeast strains were created that enabled chemical-genetic dissection of the role of different functional groups of wyebutosine that are added in a three-step post-transcriptional set of reactions. With this system, hypomodification increased Phe-specific frameshifting, with incremental changes in frameshift efficiency after specific intermediates in the progression of wyebutosine synthesis. These data combined with investigations of wild-type and hypomodified tRNA binding to ribosomes suggest that frameshift efficiency is kinetically and not thermodynamically controlled. The progressive nature of frameshift efficiency with the stage of modification is consistent with a stepwise evolution and tuning of frameshift potential. The stepwise tuning of frameshift efficiency could explain why tRNA(Phe) in some eukaryotes is not fully modified but, rather, hypomodified to capture a specific frameshift potential.

Protein multi-functionality is an emerging explanation for the complexity of higher organisms. In this regard, while aminoacyl tRNA synthetases catalyze amino acid activation for protein synthesis, some also act in pathways for inflammation, angiogenesis, and apoptosis. How multiple functions evolved and their relationship to the active site is not clear. Here structural modeling analysis, mutagenesis, and cell-based functional studies show that the potent angiostatic, natural fragment of human TrpRS associates via Trp side chains that protrude from the cognate cellular receptor VE-cadherin. Modeling indicates that (I prefer the way it was because the conclusion was reached not only by modeling, but more so by experimental studies.)VE-cadherin Trp side chains fit into the Trp-specific active site of the synthetase. Thus, specific side chains of the receptor mimic (?) amino acid substrates and expand the functionality of the active site of the synthetase. We propose that orthogonal use of the same active site may be a general way to develop multifunctionality of human tRNA synthetases and other proteins.Along with alternative splicing, post-translational modifications, and regulatory RNAs, protein multi-functionality is an emerging explanation for the complexity of higher organisms. Multi-functionality reduces the need for more protein-coding genes. In addition, expanded functions provide a way to integrate diverse biological systems, where one pathway is connected to another. Prominent among the various examples of multifunctional proteins are members of the aminoacyl tRNA synthetase (AARS) family 1, 2. While AARSs are well known for activating amino acids for protein synthesis 3, they also have a broad spectrum of expanded activities in pathways including splicing 4 ,5 , translational and transcriptional control 6-8 , pro-and anti-angiogenesis 9-14 , inflammation 15 , signaling of immune responses 16 , apoptosis 17 , and viral assembly 18 . It is these activities that may explain why there are many disease-associated tRNA synthetase mutations, especially in instances where the change has no effect on aminoacylation activity [19][20][21][22][23] . In most cases, Correspondence: xlyang@scripps.edu, Phone: (858) however, how and why tRNA synthetases were expropriated for other purposes is still not understood.Upon stimulation with the anti-proliferative cytokine interferon-γ, the 471 amino acid human tryptophanyl-tRNA synthetase (TrpRS) is up-regulated and secreted from various cell types including endothelial cells 24, 25. Natural alternative forms of human TrpRS have potent anti-angiogenic activity 9 -11 ,14 . This activity is blocked until the N-terminal domain (N-domain) is removed, either by alternative splicing to give mini-TrpRS, or by proteolysis, to give the closely similar T2-TrpRS (comprised of residues 94-471 of TrpRS). T2-TrpRS binds to the endothelial cell adherens junction molecule-vascular endothelial-cadherin (VE-cadherin) 26 -and inhibits activation of genes associated with angiogenesis ...

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