Discovery of a Gene Family Critical to Wyosine Base Formation in a Subset of Phenylalanine-specific Transfer RNAs
ABSTRACT A large number of post-transcriptional base modifications in transfer RNAs have been described (Sprinzl, M., Horn, C., Brown, M., Ioudovitch, A., and Steinberg, S. (1998) Nucleic Acids Res. 26, 148-153). These modifications enhance and expand tRNA function to increase cell viability. The intermediates and genes essential for base modifications in many instances remain unclear. An example is wyebutosine (yW), a fluorescent tricyclic modification of an invariant guanosine situated on the 3'-side of the tRNA(Phe) anticodon. Although biosynthesis of yW involves several reaction steps, only a single pathway-specific enzyme has been identified (Kalhor, H. R., Penjwini, M., and Clarke, S. (2005) Biochem. Biophys. Res. Commun. 334, 433-440). We used comparative genomics analysis to identify a cluster of orthologous groups (COG0731) of wyosine family biosynthetic proteins. Gene knock-out and complementation studies in Saccharomyces cerevisiae established a role for YPL207w, a COG0731 ortholog that encodes an 810-amino acid polypeptide. Further analysis showed the accumulation of N(1)-methylguanosine (m(1)G(37)) in tRNA from cells bearing a YPL207w deletion. A similar lack of wyosine base and build-up of m(1)G(37) is seen in certain mammalian tumor cell lines. We proposed that the 810-amino acid COG0731 polypeptide participates in converting tRNA(Phe)-m(1)G(37) to tRNA(Phe)-yW.
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- "It has been known for some time that the first step in Wye base formation is catalyzed by the m 1 G methyltransferase Trm5 (Droogmans and Grosjean 1987; Bjork et al. 2001). Recent work has shown that subsequent biosynthesis of wybutosine involves formation of the methyl imidazole ring by Tyw1 (Waas et al. 2005; Noma et al. 2006), followed by addition of the a-amino-a-carboxypropyl group of methionine by Tyw2 (Kalhor et al. 2005; Noma et al. 2006), and methylation of guanosine N3 by Tyw3 (Noma et al. 2006). Intriguingly, the last step of Wye base formation involves both methylation of the a-carboxy end group and methoxycarboxylation of the a-amino end group, with incorporation of carbon dioxide, all seemingly catalyzed by Tyw4 (Noma et al. 2006; Suzuki et al. 2009). "
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ABSTRACT: tRNA biology has come of age, revealing an unprecedented level of understanding and many unexpected discoveries along the way. This review highlights new findings on the diverse pathways of tRNA maturation, and on the formation and function of a number of modifications. Topics of special focus include the regulation of tRNA biosynthesis, quality control tRNA turnover mechanisms, widespread tRNA cleavage pathways activated in response to stress and other growth conditions, emerging evidence of signaling pathways involving tRNA and cleavage fragments, and the sophisticated intracellular tRNA trafficking that occurs during and after biosynthesis.Genes & development 09/2010; 24(17):1832-60. DOI:10.1101/gad.1956510 · 10.80 Impact Factor
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- "Remarkably the output from the PLEX search showed only the two protein families exemplified by the yeast proteins Ypl207w and Ygl050w. Experimental validation by several groups has shown that both these proteins are, indeed, involved in wybutosine biosynthesis (Kalhor et al., 2005; Noma et al., 2006; Waas et al., 2005). The output from the NMPDR and MBGD, CoGenT+ + databases were less selective. "
ABSTRACT: As the molecular adapters between codons and amino acids, transfer-RNAs are pivotal molecules of the genetic code. The coding properties of a tRNA molecule do not reside only in its primary sequence. Posttranscriptional nucleoside modifications, particularly in the anticodon loop, can modify cognate codon recognition, affect aminoacylation properties, or stabilize the codon-anticodon wobble base pairing to prevent ribosomal frameshifting. Despite a wealth of biophysical and structural knowledge of the tRNA modifications themselves, their pathways of biosynthesis had been until recently only partially characterized. This discrepancy was mainly due to the lack of obvious phenotypes for tRNA modification-deficient strains and to the difficulty of the biochemical assays used to detect tRNA modifications. However, the availability of hundreds of whole-genome sequences has allowed the identification of many of these missing tRNA-modification genes. This chapter reviews the methods that were used to identify these genes with a special emphasis on the comparative genomic approaches. Methods that link gene and function but do not rely on sequence homology will be detailed, with examples taken from the tRNA modification field.Methods in Enzymology 02/2007; 425:153-83. DOI:10.1016/S0076-6879(07)25007-4 · 2.09 Impact Factor
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ABSTRACT: Circulating lipopolysaccharide (LPS) causes a rapid transcriptional activation of its transmembrane receptor mCD14 within the circumventricular organs (CVOs), brain regions that contain a rich vascular plexus with specialized arrangements of the blood vessels. Parenchymal cells located in the anatomical boundaries of the CVOs exhibit a delayed response, which is followed by a positive signal for CD14 transcript in microglia across the brain parenchyma. The constitutive expression of the toll-like receptor 4 (TLR4) in the CVOs is likely to be a key element allowing the proinflammatory signal transduction pathways (MyD88/IRAK/NIK/NF-κB) to take place rapidly in these organs in response to circulating LPS. These results strongly suggest that the endotoxin first reaches organs devoid of the blood brain barrier (BBB) to induce the transcription of its own receptor and thereafter increases CD14 biosynthesis within parenchymal structures surrounding the CVOs and then the entire brain of severely challenged animals. Brain CD14 expression may be a key step in the transcription of proinflammatory cytokines primarily within accessible structures from the blood and subsequently through scattered parenchymal cells during severe sepsis. However, CD14 synthesis in parenchymal cells of the brain is also dependent on the production of proinflammatory cytokines. Of interest is the data that systemic injection of the bacterial endotoxin induces a strong expression of CD14 mRNA in a pattern that is closely related to the induction of tumor necrosis factor alpha (TNF-α) transcript with a rapid and delayed response. Although there is a large body of evidence that CD14 (and now TLR4) is necessary for the role of LPS on the induction of cytokine transcription from different myeloid cells, the possibility remains that the cytokine itself acts as an autocrine and paracrine factor to up regulate the LPS receptor. The binding of TNF to its type I receptor (p55) leads to the activation and translocation of p50/65 NF-κB into the nucleus, which seems a key player in activating CD14 transcription in the CNS. Central injection of recombinant rat TNF-α causes a robust expression of the genes encoding IκBα, TNF-α and CD14 in microglial cells of the brain parenchyma. The time-related induction of these transcripts suggested a potential role of NF-κB in mediating TNF-induced transcriptional activation of the LPS receptor. Systemic injection with the endotoxin LPS provoked a similar microglial activation that was prevented in inhibiting the biological activity of the proinflammatory cytokine in the CNS. Together these data provide the evidence that centrally-produced TNF-α plays an essential autocrine/paracrine role in triggering parenchymal microglial cells during severe endotoxemia. These events may be determinant for orchestrating the neuroinflammatory responses that take place in a well coordinated manner to activate the resident phagocytic population of cells in the brain. The physiological outcomes of this innate immune response of the CNS are likely to include a rapid elimination of LPS particles via an increased opsonic activity of the transmembrane CD14 receptor to prevent potential detrimental consequences on neuronal elements during blood sepsis.