Molecular basis of cobalamin-dependent RNA modification

Queuosine (Q) was discovered in the wobble position of a transfer RNA (tRNA) 47 years ago, yet the final biosynthetic enzyme responsible for Q-maturation, epoxyqueuosine (oQ) reductase (QueG), was only recently identified. QueG is a cobalamin (Cbl)-dependent, [4Fe-4S] cluster-containing protein that produces the hypermodified nucleoside Q in situ on four tRNAs. To understand how QueG is able to perform epoxide reduction, an unprecedented reaction for a Cbl-dependent enzyme, we have determined a series of high resolution structures of QueG from Bacillus subtilis. Our structure of QueG bound to a tRNA[superscript Tyr] anticodon stem loop shows how this enzyme uses a HEAT-like domain to recognize the appropriate anticodons and position the hypermodified nucleoside into the enzyme active site. We find Q bound directly above the Cbl, consistent with a reaction mechanism that involves the formation of a covalent Cbl-tRNA intermediate. Using protein film electrochemistry, we show that two [4Fe-4S] clusters adjacent to the Cbl have redox potentials in the range expected for Cbl reduction, suggesting how Cbl can be activated for nucleophilic attack on oQ. Together, these structural and electrochemical data inform our understanding of Cbl dependent nucleic acid modification.National Science Foundation (U.S.) (MCB 1122977)National Institutes of Health (U.S.) (GM72623 S01, GM120283, and GM17151

C21orf57 is a human homologue of bacterial YbeY proteins

The product of the human C21orf57 (huYBEY) gene is predicted to be a homologue of the highly conserved YbeY proteins found in nearly all bacteria. We show that, like its bacterial and chloroplast counterparts, the HuYbeY protein is an RNase and that it retains sufficient function in common with bacterial YbeY proteins to partially suppress numerous aspects of the complex phenotype of an Escherichia coli ΔybeY mutant. Expression of HuYbeY in Saccharomyces cerevisiae, which lacks a YbeY homologue, results in a severe growth phenotype. This observation suggests that the function of HuYbeY in human cells is likely regulated through specific interactions with partner proteins similarly to the way YbeY is regulated in bacteria.National Institutes of Health (U.S.) (Grant GM31010)National Institutes of Health (U.S.) (Grant GM17151

Identification and codon reading properties of 5-cyanomethyl uridine, a new modified nucleoside found in the anticodon wobble position of mutant haloarchaeal isoleucine tRNAs

Most archaea and bacteria use a modified C in the anticodon wobble position of isoleucine tRNA to base pair with A but not with G of the mRNA. This allows the tRNA to read the isoleucine codon AUA without also reading the methionine codon AUG. To understand why a modified C, and not U or modified U, is used to base pair with A, we mutated the C34 in the anticodon of Haloarcula marismortui isoleucine tRNA (tRNA2Ile) to U, expressed the mutant tRNA in Haloferax volcanii, and purified and analyzed the tRNA. Ribosome binding experiments show that although the wild-type tRNA2Ile binds exclusively to the isoleucine codon AUA, the mutant tRNA binds not only to AUA but also to AUU, another isoleucine codon, and to AUG, a methionine codon. The G34 to U mutant in the anticodon of another H. marismortui isoleucine tRNA species showed similar codon binding properties. Binding of the mutant tRNA to AUG could lead to misreading of the AUG codon and insertion of isoleucine in place of methionine. This result would explain why most archaea and bacteria do not normally use U or a modified U in the anticodon wobble position of isoleucine tRNA for reading the codon AUA. Biochemical and mass spectrometric analyses of the mutant tRNAs have led to the discovery of a new modified nucleoside, 5-cyanomethyl U in the anticodon wobble position of the mutant tRNAs. 5-Cyanomethyl U is present in total tRNAs from euryarchaea but not in crenarchaea, eubacteria, or eukaryotes.National Institutes of Health (U.S.) (GM17151)National Institutes of Health (U.S.) (GM22854)National Institutes of Health (U.S.) (ES017010)Singapore-MIT Alliance for Research and TechnologySingapore. National Research FoundationUnited States. Dept. of Energy (DE-FG36-08GO88055

Impaired protein translation in Drosophila models for Charcot–Marie–Tooth neuropathy caused by mutant tRNA synthetases

Dominant mutations in five tRNA synthetases cause Charcot–Marie–Tooth (CMT) neuropathy, suggesting that altered aminoacylation function underlies the disease. However, previous studies showed that loss of aminoacylation activity is not required to cause CMT. Here we present a Drosophila model for CMT with mutations in glycyl-tRNA synthetase (GARS). Expression of three CMT-mutant GARS proteins induces defects in motor performance and motor and sensory neuron morphology, and shortens lifespan. Mutant GARS proteins display normal subcellular localization but markedly reduce global protein synthesis in motor and sensory neurons, or when ubiquitously expressed in adults, as revealed by FUNCAT and BONCAT. Translational slowdown is not attributable to altered tRNA[superscript Gly] aminoacylation, and cannot be rescued by Drosophila Gars overexpression, indicating a gain-of-toxic-function mechanism. Expression of CMT-mutant tyrosyl-tRNA synthetase also impairs translation, suggesting a common pathogenic mechanism. Finally, genetic reduction of translation is sufficient to induce CMT-like phenotypes, indicating a causal contribution of translational slowdown to CMT.National Institutes of Health (U.S.) (Grant GM17151

Nonsense suppression in archaea

Bacterial strains carrying nonsense suppressor tRNA genes played a crucial role in early work on bacterial and bacterial viral genetics. In eukaryotes as well, suppressor tRNAs have played important roles in the genetic analysis of yeast and worms. Surprisingly, little is known about genetic suppression in archaea, and there has been no characterization of suppressor tRNAs or identification of nonsense mutations in any of the archaeal genes. Here, we show, using the β-gal gene as a reporter, that amber, ochre, and opal suppressors derived from the serine and tyrosine tRNAs of the archaeon Haloferax volcanii are active in suppression of their corresponding stop codons. Using a promoter for tRNA expression regulated by tryptophan, we also show inducible and regulatable suppression of all three stop codons in H. volcanii. Additionally, transformation of a ΔpyrE2 H. volcanii strain with plasmids carrying the genes for a pyrE2 amber mutant and the serine amber suppressor tRNA yielded transformants that grow on agar plates lacking uracil. Thus, an auxotrophic amber mutation in the pyrE2 gene can be complemented by expression of the amber suppressor tRNA. These results pave the way for generating archaeal strains carrying inducible suppressor tRNA genes on the chromosome and their use in archaeal and archaeviral genetics. We also provide possible explanations for why suppressor tRNAs have not been identified in archaea.National Institutes of Health (U.S.) (Grant GM17151

Comparison of two methods to measure macular pigment optical density in healthy subjects

PURPOSE: To evaluate and compare macular pigment optical density (MPOD) measurements obtained using the modified Heidelberg Retina Angiograph (HRA) and the Visucam 200. METHODS: Healthy young subjects were included in this prospective study. MPOD was measured with the modified HRA at 0 degrees and 0.5 degrees , 1 degrees , 2 degrees , and 6 degrees eccentricities from the fovea. The parameters obtained with the Visucam 200 (maximum, mean, area, and volume) were recorded the same day on the same subjects. Intraclass correlation coefficients (ICCs) and correlation coefficients were used to evaluate the agreement between the two devices. The repeatability and the reproducibility of each method were also assessed. RESULTS: Sixty-seven subjects were included whose median (interquartile ratio) age was 25 years (range, 23-30 years). The MPODs as measured with the modified HRA were higher than those measured with the Visucam 200 (P < 0.0001). The ICCs were low, ranging from 0.020 to 0.188. The correlation coefficients between the two methods were very low and ranged from 0.05 to 0.22. Repeatability and reproducibility were good with both methods, with ICCs ranging from 0.697 to 0.923. CONCLUSIONS: Agreement between the modified HRA and the Visucam in measuring MPOD was rather low. These results suggest that the two methods are not interchangeable. Before using the Visucam 200 in clinical and research setting, further evaluation seems mandatory (http://ansm.sante.fr/ number, 2009-A00448-49)

The structural basis for specific decoding of AUA by isoleucine tRNA on the ribosome

Decoding of the AUA isoleucine codon in bacteria and archaea requires modification of a C in the anticodon wobble position of the isoleucine tRNA. Here, we report the crystal structure of the archaeal tRNA2Ile, which contains the modification agmatidine in its anticodon, in complex with the AUA codon on the 70S ribosome. The structure illustrates how agmatidine confers codon specificity for AUA over AUG.Medical Research Council (Great Britain) (grant U105184332)Agouron InstituteWellcome Trust (London, England)Louis-Jeantet FoundationNational Institutes of Health (U.S.) (grant GM17151)University of Cambridge (Gates Cambridge Scholarship)Peterhouse (University of Cambridge)Boehringer Ingelheim Fond

Life without tRNA[superscript Ile]-lysidine synthetase: translation of the isoleucine codon AUA in Bacillus subtilis lacking the canonical tRNA[Ile over 2]

Translation of the isoleucine codon AUA in most prokaryotes requires a modified C (lysidine or agmatidine) at the wobble position of tRNA[Ile over 2] to base pair specifically with the A of the AUA codon but not with the G of AUG. Recently, a Bacillus subtilis strain was isolated in which the essential gene encoding tRNA[superscript Ile]-lysidine synthetase was deleted for the first time. In such a strain, C34 at the wobble position of tRNA[Ile over 2] is expected to remain unmodified and cells depend on a mutant suppressor tRNA derived from tRNA[Ile over 1], in which G34 has been changed to U34. An important question, therefore, is how U34 base pairs with A without also base pairing with G. Here, we show (i) that unlike U34 at the wobble position of all B. subtilis tRNAs of known sequence, U34 in the mutant tRNA is not modified, and (ii) that the mutant tRNA binds strongly to the AUA codon on B. subtilis ribosomes but only weakly to AUG. These in vitro data explain why the suppressor strain displays only a low level of misreading AUG codons in vivo and, as shown here, grows at a rate comparable to that of the wild-type strain.National Institutes of Health (U.S.) (GM17151

The G Protein-Coupled Bile Acid Receptor TGR5 (Gpbar1) Modulates Endothelin-1 Signaling in Liver

TGR5 (Gpbar1) is a G protein-coupled receptor responsive to bile acids (BAs), which is expressed in different non-parenchymal cells of the liver, including biliary epithelial cells, liver-resident macrophages, sinusoidal endothelial cells (LSECs), and activated hepatic stellate cells (HSCs). Mice with targeted deletion of TGR5 are more susceptible towards cholestatic liver injury induced by cholic acid-feeding and bile duct ligation, resulting in a reduced proliferative response and increased liver injury. Conjugated lithocholic acid (LCA) represents the most potent TGR5 BA ligand and LCA-feeding has been used as a model to rapidly induce severe cholestatic liver injury in mice. Thus, TGR5 knockout (KO) mice and wildtype (WT) littermates were fed a diet supplemented with 1% LCA for 84 h. Liver injury and gene expression changes induced by the LCA diet revealed an enrichment of pathways associated with inflammation, proliferation, and matrix remodeling. Knockout of TGR5 in mice caused upregulation of endothelin-1 (ET-1) expression in the livers. Analysis of TGR5-dependent ET-1 signaling in isolated LSECs and HSCs demonstrated that TGR5 activation reduces ET-1 expression and secretion from LSECs and triggers internalization of the ET-1 receptor in HSCs, dampening ET-1 responsiveness. Thus, we identified two independent mechanisms by which TGR5 inhibits ET-1 signaling and modulates portal pressure

Essentiality of threonylcarbamoyladenosine (t6A), a universal tRNA modification, in bacteria

Threonylcarbamoyladenosine (t[superscript 6]A) is a modified nucleoside universally conserved in tRNAs in all three kingdoms of life. The recently discovered genes for t6A synthesis, including tsaC and tsaD, are essential in model prokaryotes but not essential in yeast. These genes had been identified as antibacterial targets even before their functions were known. However, the molecular basis for this prokaryotic-specific essentiality has remained a mystery. Here, we show that t[superscript 6]A is a strong positive determinant for aminoacylation of tRNA by bacterial-type but not by eukaryotic-type isoleucyl-tRNA synthetases and might also be a determinant for the essential enzyme tRNA[superscript Ile]-lysidine synthetase. We confirm that t6A is essential in Escherichia coli and a survey of genomewide essentiality studies shows that genes for t[superscrip 6]A synthesis are essential in most prokaryotes. This essentiality phenotype is not universal in Bacteria as t[superscript 6]A is dispensable in Deinococcus radiodurans, Thermus thermophilus, Synechocystis PCC6803 and Streptococcus mutans. Proteomic analysis of t6A- D. radiodurans strains revealed an induction of the proteotoxic stress response and identified genes whose translation is most affected by the absence of t[superscript 6]A in tRNAs. Thus, although t[superscript 6]A is universally conserved in tRNAs, its role in translation might vary greatly between organisms.National Institutes of Health (U.S.) (Grant R01 GM17151