Quality control of protein synthesis in the early elongation stage.

In the early stage of bacterial translation, peptidyl-tRNAs frequently dissociate from the ribosome (pep-tRNA drop-off) and are recycled by peptidyl-tRNA hydrolase. Here, we establish a highly sensitive method for profiling of pep-tRNAs using mass spectrometry, and successfully detect a large number of nascent peptides from pep-tRNAs accumulated in Escherichia coli pthts strain. Based on molecular mass analysis, we found about 20% of the peptides bear single amino-acid substitutions of the N-terminal sequences of E. coli ORFs. Detailed analysis of individual pep-tRNAs and reporter assay revealed that most of the substitutions take place at the C-terminal drop-off site and that the miscoded pep-tRNAs rarely participate in the next round of elongation but dissociate from the ribosome. These findings suggest that pep-tRNA drop-off is an active mechanism by which the ribosome rejects miscoded pep-tRNAs in the early elongation, thereby contributing to quality control of protein synthesis after peptide bond formation.

Advanced Boron Neutron Capture Therapy Targeting Cancer Stem Cells by Selective Induction of LAT1 Overexpression.

This study conducted fundamental research to develop a more effective BNCT targeting cancer stem cells. We constructed plasmids that induced the overexpression of L-type amino acid transporter 1 (LAT1) tagged with tdTomato on the cytoplasmic membranes of CD133 expressing cancer cells. After transfection of the plasmids into a glioblastoma cell line (T98G), several clones overexpressing LAT1-tdTomato in the hypoxic microenvironment of the spheroids formed from each clone were obtained. Confocal laser microscopic observation confirmed that signals from LAT1-tdTomato overlapped with immunofluorescence signals from the second antibody binding to CD133 in the hypoxic microenvironment of the spheroids. As CD133-positive cells in the hypoxic microenvironment of T98G spheroids have cancer stem cell characteristics, LAT1 seems to be selectively overexpressed in cancer stem cell-like cells. An RI tracer method showed that cells overexpressing LAT1-tdTomato in the hypoxic microenvironment of spheroids incorporate 14C-BPA much more than cells that do not overexpress LAT1-tdTomato. Neutron radiation experiments showed a more significant regression in spheroids formed with clones than in spheroids formed with parental cells when spheroids were treated with 10BPA. These results suggest that BNCT combined with gene therapy targeting cancer stem cells is more effective in glioblastoma therapy.

The landscape of translational stall sites in bacteria revealed by monosome and disome profiling.

Ribosome pauses are associated with various cotranslational events and determine the fate of mRNAs and proteins. Thus, the identification of precise pause sites across the transcriptome is desirable; however, the landscape of ribosome pauses in bacteria remains ambiguous. Here, we harness monosome and disome (or collided ribosome) profiling strategies to survey ribosome pause sites in Escherichia coli Compared to eukaryotes, ribosome collisions in bacteria showed remarkable differences: a low frequency of disomes at stop codons, collisions occurring immediately after 70S assembly on start codons, and shorter queues of ribosomes trailing upstream. The pause sites corresponded with the biochemical validation by integrated nascent chain profiling (iNP) to detect polypeptidyl-tRNA, an elongation intermediate. Moreover, the subset of those sites showed puromycin resistance, presenting slow peptidyl transfer. Among the identified sites, the ribosome pause at Asn586 of ycbZ was validated by biochemical reporter assay, tRNA sequencing (tRNA-seq), and cryo-electron microscopy (cryo-EM) experiments. Our results provide a useful resource for ribosome stalling sites in bacteria.

Ribosome stalling caused by the Argonaute-microRNA-SGS3 complex regulates the production of secondary siRNAs in plants.

The path of ribosomes on mRNAs can be impeded by various obstacles. One such example is halting of ribosome movement by microRNAs, but the exact mechanism and physiological role remain unclear. Here, we find that ribosome stalling caused by the Argonaute-microRNA-SGS3 complex regulates production of secondary small interfering RNAs (siRNAs) in plants. We show that the double-stranded RNA-binding protein SGS3 interacts directly with the 3' end of the microRNA in an Argonaute protein, resulting in ribosome stalling. Importantly, microRNA-mediated ribosome stalling correlates positively with efficient production of secondary siRNAs from target mRNAs. Our results illustrate a role of paused ribosomes in regulation of small RNA function that may have broad biological implications across the plant kingdom.

The Pentatricopeptide Repeat Protein PGR3 Is Required for the Translation of petL and ndhG by Binding Their 5' UTRs.

PGR3 is a P-class pentatricopeptide repeat (PPR) protein required for the stabilization of petL operon RNA and the translation of the petL gene in plastids. Irrespective of its important roles in plastids, key questions have remained unanswered, including how PGR3 protein promotes translation and which plastid mRNA PGR3 activates the translation. Here, we show that PGR3 facilitates the translation from ndhG, in addition to petL, through binding to their 5' untranslated regions (UTRs). Ribosome profiling and RNA sequencing in pgr3 mutants revealed that translation from petL and ndhG was specifically suppressed. Harnessing small RNA fragments protected by PPR proteins in vivo, we probed the PGR3 recruitment to the 5' UTRs of petL and ndhG. The putative PGR3-bound RNA segments per se repress the translation possibly with a strong secondary structure and thereby block ribosomes' access. However, the PGR3 binding antagonizes the effects and facilitates the protein synthesis from petL and ndhG in vitro. The prediction of the 3-dimensional structure of PGR3 suggests that the 26th PPR motif plays important roles in target RNA binding. Our data show the specificity of a plastidic RNA-binding protein and provide a mechanistic insight into translational control.

Translational Landscape of Protein-Coding and Non-Protein-Coding RNAs upon Light Exposure in Arabidopsis.

Light is one of the most essential environmental clues for plant growth and morphogenesis. Exposure to blue monochromatic light from darkness is a turning point for plant biological activity, and as a result dramatic changes in gene expression occur. To understand the translational impacts of blue light, we have performed ribosome profiling analysis and called translated open reading frames (ORFs) de novo within not only mRNAs but also non-coding RNAs (ncRNAs). Translation efficiency of 3,823 protein-coding ORFs, such as nuclear chloroplast-related genes, was up-regulated by blue light exposure. Moreover, the translational activation of the microRNA biogenesis-related genes, DCL1 and HYL1, was induced by blue light. Considering the 3-nucleotide codon periodicity of ribosome footprints, a few hundred short ORFs lying on ncRNAs and upstream ORFs (uORFs) on mRNAs were found that had differential translation status between blue light and dark. uORFs are known to have a negative effect on the expression of the main ORFs (mORFs) on the same mRNAs. Our analysis suggests that the translation of uORFs is likely to be more stimulated than that of the corresponding mORFs, and uORF-mediated translational repression of the mORFs in five genes was alleviated by blue light exposure. With data-based annotation of the ORFs, our analysis provides insights into the translatome in response to environmental changes, such as those involving light.

The Plant Translatome Surveyed by Ribosome Profiling.

Although transcriptome changes have long been recognized as a mechanism to induce tentative substitution of expressed genes in diverse biological processes in plants, the regulation of translation-the final step of the central dogma of molecular biology-emerged as an alternative and prominent layer in defining the output of genes. Despite these demands, the genome-wide analysis of protein synthesis has posed technical challenges, resulting in the plant translatome being poorly understood. The development of ribosome profiling promises to address the hidden aspects of translation, and its application to plants is revolutionizing our knowledge of the translatome. This review outlines the array of recent findings provided by ribosome profiling and illustrates the power of the versatile technique in green organisms.

Transcripts from downstream alternative transcription start sites evade uORF-mediated inhibition of gene expression in Arabidopsis.

Plants adapt to alterations in light conditions by controlling their gene expression profiles. Expression of light-inducible genes is transcriptionally induced by transcription factors such as HY5. However, few detailed analyses have been carried out on the control of transcription start sites (TSSs). Of the various wavelengths of light, it is blue light (BL) that regulates physiological responses such as hypocotyl elongation and flowering time. To understand how gene expression is controlled not only by transcript abundance but also by TSS selection, we examined genome-wide TSS profiles in Arabidopsis seedlings after exposure to BL irradiation following initial growth in the dark. Thousands of genes use multiple TSSs, and some transcripts have upstream ORFs (uORFs) that take precedence over the main ORF (mORF) encoding proteins. The uORFs often function as translation inhibitors of the mORF or as triggers of nonsense-mediated mRNA decay (NMD). Transcription from TSSs located downstream of the uORFs in 220 genes is enhanced by BL exposure. This type of regulation is found in HY5 and HYH, major regulators of light-dependent gene expression. Translation efficiencies of the genes showing enhanced usage of these TSSs increased upon BL exposure. We also show that transcripts from TSSs upstream of uORFs in 45 of the 220 genes, including HY5, accumulated in a mutant of NMD. These results suggest that BL controls gene expression not only by enhancing transcriptions but also by choosing the TSS, and transcripts from downstream TSSs evade uORF-mediated inhibition to ensure high expression of light-regulated genes.

Aspergillus oryzae pathways that convert phenylalanine into the flavor volatile 2-phenylethanol.

The filamentous fungus Aspergillus oryzae RIB40 produced 2-phenylethanol (PE) when cultured in minimum medium containing l-phenylalanine as a sole source of nitrogen. The fungus accumulated less PE in the absence of l-phenylalanine, indicating that it converted l-phenylalanine to PE. The PE production associated with fungal glucose consumption was repressed by exogenous ammonium, indicating that nitrogen-metabolite repression controls the pathway that produces PE. We identified the A. oryzae ppdA gene that is expressed at high levels in the presence of exogenous l-phenylalanine and its encoded protein was an active phenylpyruvate decarboxylase. The fungal genome encodes predicted aminotransferases of phenylalanine and PE dehydrogenases, which, together with PpdA, are likely to constitute an Erlich pathway similar to that in Saccharomyces cerevisiae that produces PE. We also identified an A. oryzae aromatic amino acid decarboxylase (AadA) that converted l-phenylalanine to phenylethylamine (PEA), and phenylalanine-inducible PEA oxidase activity in fungal cell extracts, and found that both constitute an alternative pathway through which PEA generates PE. Incubating fungal cultures with l-[(2)H8] phenylalanine to distinguish PE produced by these pathways, indicated that the fungus produced PE by both pathways, but to a greater extent by the Erlich pathway. Gene disruption of ppdA and aadA showed that both pathways participate in the fungal conversion of l-phenylalanine to PE.

Achromobacter denitrificans strain YD35 pyruvate dehydrogenase controls NADH production to allow tolerance to extremely high nitrite levels.

We identified the extremely nitrite-tolerant bacterium Achromobacter denitrificans YD35 that can grow in complex medium containing 100 mM nitrite (NO2(-)) under aerobic conditions. Nitrite induced global proteomic changes and upregulated tricarboxylate (TCA) cycle enzymes as well as antioxidant proteins in YD35. Transposon mutagenesis generated NO2(-)-hypersensitive mutants of YD35 that had mutations at genes for aconitate hydratase and α-ketoglutarate dehydrogenase in the TCA cycle and a pyruvate dehydrogenase (Pdh) E1 component, indicating the importance of TCA cycle metabolism to NO2(-) tolerance. A mutant in which the pdh gene cluster was disrupted (Δpdh mutant) could not grow in the presence of 100 mM NO2(-). Nitrite decreased the cellular NADH/NAD(+) ratio and the cellular ATP level. These defects were more severe in the Δpdh mutant, indicating that Pdh contributes to upregulating cellular NADH and ATP and NO2(-)-tolerant growth. Exogenous acetate, which generates acetyl coenzyme A and then is metabolized by the TCA cycle, compensated for these defects caused by disruption of the pdh gene cluster and those caused by NO2(-). These findings demonstrate a link between NO2(-) tolerance and pyruvate/acetate metabolism through the TCA cycle. The TCA cycle mechanism in YD35 enhances NADH production, and we consider that this contributes to a novel NO2(-)-tolerating mechanism in this strain.

NO-inducible nitrosothionein mediates NO removal in tandem with thioredoxin.

Nitric oxide (NO) is a toxic reactive nitrogen species that induces microbial adaption mechanisms. Screening a genomic DNA library identified a new gene, ntpA, that conferred growth tolerance upon Aspergillus nidulans against exogenous NO. The gene encoded a cysteine-rich 23-amino-acid peptide that reacted with NO and S-nitrosoglutathione to generate an S-nitrosated peptide. Disrupting ntpA increased amounts of cellular S-nitrosothiol and NO susceptibility. Thioredoxin and its reductase denitrosated the S-nitrosated peptide, decreased cellular S-nitrosothiol and conferred tolerance against NO, indicating peptide-mediated catalytic NO removal. The peptide binds copper(I) in vitro but is dispensable for metal tolerance in vivo. NO but not metal ions induced production of the peptide and ntpA transcripts. We discovered that the thionein family of peptides has NO-related functions and propose that the new peptide be named NO-inducible nitrosothionein (iNT). The ubiquitous distribution of iNT-like polypeptides constitutes a potent NO-detoxifying mechanism that is conserved among various organisms.

Microbial monomers custom-synthesized to build true bio-derived aromatic polymers.

Aromatic polymers include novel and extant functional materials although none has been produced from biotic building blocks derived from primary biomass glucose. Here we screened microbial aromatic metabolites, engineered bacterial metabolism and fermented the aromatic lactic acid derivative ß-phenyllactic acid (PhLA). We expressed the Wickerhamia fluorescens gene (pprA) encoding a phenylpyruvate reductase in Escherichia coli strains producing high levels of phenylalanine, and fermented optically pure (>99.9 %) D-PhLA. Replacing pprA with bacterial ldhA encoding lactate dehydrogenase generated L-PhLA, indicating that the produced enzymes converted phenylpyruvate, which is an intermediate of phenylalanine synthesis, to these chiral PhLAs. Glucose was converted under optimized fermentation conditions to yield 29 g/l D-PhLA, which was purified from fermentation broth. The product satisfied the laboratory-scale chemical synthesis of poly(D-PhLA) with M w 28,000 and allowed initial physiochemical characterization. Poly(D-PhLA) absorbed near ultraviolet light, and has the same potential as all other biomass-derived aromatic bioplastics of phenylated derivatives of poly(lactic acid). This approach to screening and fermenting aromatic monomers from glucose exploits a new era of bio-based aromatic polymer design and will contribute to petroleum conservation and carbon dioxide fixation.

Hydrolase controls cellular NAD, sirtuin, and secondary metabolites.

Cellular levels of NAD(+) and NADH are thought to be controlled by de novo and salvage mechanisms, although evidence has not yet indicated that they are regulated by NAD(+) degradation. Here we show that the conserved nudix hydrolase isozyme NdxA hydrolyzes and decreases cellular NAD(+) and NADH in Aspergillus nidulans. The NdxA-deficient fungus accumulated more NAD(+) during the stationary growth phase, indicating that NdxA maintains cellular NAD(+)/NADH homeostasis. The deficient strain also generated less of the secondary metabolites sterigmatocystin and penicillin G and of their gene transcripts than did the wild type. These defects were associated with a reduction in acetylated histone H4 on the gene promoters of aflR and ipnA that are involved in synthesizing secondary metabolites. Thus, NdxA increases acetylation levels of histone H4. We discovered that the novel fungal sirtuin isozyme SirA uses NAD(+) as a cosubstrate to deacetylate the lysine 16 residue of histone H4 on the gene promoter and represses gene expression. The impaired acetylation of histone and secondary metabolite synthesis in the NdxA-deficient strain were restored by eliminating functional SirA, indicating that SirA mediates NdxA-dependent regulation. These results indicated that NdxA controls total levels of NAD(+)/NADH and negatively regulates sirtuin function and chromatin structure.

Novel fungal phenylpyruvate reductase belongs to d-isomer-specific 2-hydroxyacid dehydrogenase family.

We discovered the phenyllactate (PLA)-producing fungal strain Wickerhamia fluorescens TK1 and purified phenylpyruvate reductase (PPR) from fungal cell-free extracts. The PPR used both NADPH and NADH as cofactors with more preference for the former. The enzyme reaction as well as the fungal culture produced optically active d-PLA. The gene for the PPR (pprA) was cloned and expressed in Escherichia coli cells. Purified preparations of both native and recombinant PPR used hydroxyphenylpyruvate, glyoxylate and hydroxypyruvate as substrates but not pyruvate, oxaloacetate or benzoylformate. The predicted PPR protein had sequence similarity to proteins in the d-isomer-specific 2-hydroxyacid dehydrogenase family. Phylogenetic analyses indicated that the predicted PPR protein together with fungal predicted proteins constitutes a novel group of glyoxylate/hydroxypyruvate reductases. The fungus efficiently converted phenylalanine and phenylpyruvate to d-PLA. These compounds up-regulated the transcription of pprA, suggesting that it plays a role in fungal phenylalanine metabolism.

Effect of mixed organic substrate on alpha-tocopherol production by Euglena gracilis in photoheterotrophic culture.

Effects of organic carbon sources on cell growth and alpha-tocopherol productivity in wild and chloroplast-deficient W14ZUL strains of Euglena gracilis under photoheterotrophic culture were investigated. In both strains, the increase in cell growth was particularly high when glucose was added as the sole organic carbon source. On the other hand, alpha-tocopherol production per dry cell weight was enhanced by adding ethanol. Ethanol addition also increased the chlorophyll concentration in wild strain and mitochondria activity in W14ZUL strain. For effective alpha-tocopherol production, the effects of mixture of glucose and ethanol were investigated. The results showed that, when a mixture of glucose (6 g/l) and ethanol (4 g/l) was used, alpha-tocopherol productivity per culture broth was 3.89 x 10(-2) mg l(-1) h(-1), which was higher than the value obtained without addition of organic carbon source (0.92 x 10(-2) mg l(-1) h(-1)). In addition, under fed-batch cultivation using an internally illuminated photobioreactor, the alpha-tocopherol production per culture broth was 23.43 mg/l, giving a productivity of 16.27 x 10(-2) mg l(-1) h(-1).

Synthesis of the new mannosidase inhibitors, diversity-oriented 5-substituted swainsonine analogues, via stereoselective Mannich reaction.

5alpha-substituted swainsonine analogues were synthesized by Mannich reaction of an in situ generated (-)-swainsonine iminium ion intermediate. 5alpha-substituted swainsonine analogues were epimerized to their 5beta-isomers in protic solvent. [reaction: see text]

First total synthesis of artepillin C established by o,o'-diprenylation of p-halophenols in water.

We have demonstrated that prenylation of p-halophenols was dependent on the solvent effect and succeeded in o,o'-diprenylation of p-halophenols in water. Following the Mizoroki-Heck coupling of the diprenyl-p-iodophenol 3c with methyl acrylate and then hydrolysis, we first synthesized artepillin C [3-(4-hydroxy-3,5-di(3-methyl-2-butenyl)phenyl)-2(E)-propenoic acid] (1), which is a biologically active constituent of propolis. These reactions may be applicable to the synthesis of various useful natural products such as 2,4,6-trisubstituted phenol derivatives.