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Glutamine analogs are potent suppressors of general glutamine metabolism with anti-cancer activity. 6-diazo-5-oxo-L-norleucine (DON) is an orally available glutamine analog which has been recently improved by structural modification for cancer treatment. Here, we explored the chemogenomic landscape of DON sensitivity using budding yeast as model organism. We identify evolutionarily conserved proteins that mediate cell resistance to glutamine analogs, namely Ura8CTPS1/2, Hpt1HPRT1, Mec1ATR, Rad53CHK1/CHK2 and Rtg1. We describe a function of Ura8 as inducible CTP synthase responding to inhibition of glutamine metabolism and propose a model for its regulation by CTP levels and Nrd1-dependent transcription termination at a cryptic unstable transcript. Disruption of the inducible CTP synthase under DON exposure hyper-activates the Mec1-Rad53 DNA damage response (DDR) pathway, which prevents chromosome breakage. Simultaneous inhibition of CTP synthase and Mec1 kinase synergistically sensitizes cells to DON, whereas CTP synthase over-expression hampers DDR mutant sensitivity. Using genome-wide suppressor screening, we identify factors promoting DON-induced CTP depletion (TORC1, glutamine transporter) and DNA breakage in DDR mutants. Together, our results identify CTP regulation and the Mec1-Rad53 DDR axis as key glutamine analog response pathways, and provide a rationale for the combined targeting of glutamine and CTP metabolism in DDR-deficient cancers. Author summary: Cancer cell proliferation is supported by high metabolic activity. Targeting metabolic pathways is therefore a strategy to suppress cancer cell growth and survival. Glutamine is a key metabolite that supports a plethora of anabolic, growth-promoting reactions in the cell. Therefore, the use of small molecules that block glutamine-dependent reactions has been extensively investigated in cancer therapy. Knowledge about the pathways that influence sensitivity towards glutamine metabolism inhibitors would help to tailor the use of such glutamine-targeting therapies. In this study, we use budding yeast as model system to identify the pathways that mediate or restrict the toxicity of a representative inhibitor of glutamine metabolism, the glutamine analog 6-diazo-5-oxo-L-norleucine (DON). We describe a response mechanism mediated by an inducible CTP synthase that promotes nucleotide homeostasis during DON exposure to prevent DNA breaks. Moreover, we show that combined inhibition of the inducible CTP synthase and DNA damage response enhances DON toxicity, pointing out a potential therapeutic application in cancers with defective DNA damage response. [ABSTRACT FROM AUTHOR]

A common core RNP structure has been found to be shared by the spliceosomal U4 snRNP and the small nucleolar box C/D RNPs. The box CD motif binds Snu13p/15.5kD in vitro. It may be that the U4 snRNP (chaperone for U6) and the box CD snoRNPOs have evolved from a common ancestral RNP.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/S0092-8674(00)80829-6 Byline: Mutsuhito Ohno (1), Alexandra Segref (1), Angela Bachi (1), Matthias Wilm (1), Iain W Mattaj (1) Abstract: In metazoa, assembly of spliceosomal U snRNPs requires nuclear export of U snRNA precursors. Export depends upon the RNA cap structure, nuclear cap-binding complex (CBC), the export receptor CRM1/Xpo1, and RanGTP. These components are however insufficient to support U snRNA export. We identify PHAX (phosphorylated adaptor for RNA export) as the additonal factor required for U snRNA export complex assembly in vitro. In vivo, PHAX is required for U snRNA export but not for CRM1-mediated export in general. PHAX is phosphorylated in the nucleus and then exported with RNA to the cytoplasm, where it is dephosphorylated. PHAX phosphorylation is essential for export complex assembly while its dephosphorylation causes export complex disassembly. The compartmentalized PHAX phosphorylation cycle can contribute to the directionality of export. Author Affiliation: (1) European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117 Heidelberg, Germany Article History: Received 6 December 1999; Revised 2 February 2000

Abstract Background All studies aimed at understanding complex molecular changes occurring at synapses face the problem of how a complete view of the synaptic proteome and of its changes can be efficiently met. This is highly desirable when synaptic plasticity processes are analyzed since the structure and the biochemistry of neurons and synapses get completely reshaped. Because most molecular studies of synapses are nowadays mainly or at least in part based on protein extracts from neuronal cultures, this is not a feasible option: these simplified versions of the brain tissue on one hand provide an homogeneous pure population of neurons but on the other yield only tiny amounts of proteins, many orders of magnitude smaller than conventional brain tissue. As a way to overcome this limitation and to find a simple way to screen for protein changes at cultured synapses, we have produced and characterized two dimensional electrophoresis (2DE) maps of the synaptic proteome of CA3-CA1 hippocampal neurons in culture. Results To obtain 2D maps, hippocampal cultures were mass produced and after synaptic maturation, proteins were extracted following subfractionation procedures and separated by 2D gel electrophoresis. Similar maps were obtained for the crude cytosol of cultured neurons and for synaptosomes purified from CA3-CA1 hippocampal tissue. To efficiently compare these different maps some clearly identifiable reference points were molecularly identified by mass spectrometry and immunolabeling methods. This information was used to run a differential analysis and establish homologies and dissimilarities in these 2D protein profiles. Conclusion Because reproducible fingerprints of cultured synapses were clearly obtained, we believe that our mapping effort could represent a simple tool to screen for protein expression and/or protein localization changes in CA3-CA1 hippocampal neurons following plasticity.