Functional Consequences of Shifting Transcript Boundaries in Glucose Starvation.

Glucose is a major source of carbon and essential for the survival of many organisms, ranging from yeast to human. A sudden 60-fold reduction of glucose in exponentially growing fission yeast induces transcriptome-wide changes in gene expression. This regulation is multilayered, and the boundaries of transcripts are known to vary, with functional consequences at the protein level. By combining direct RNA sequencing with 5'-CAGE and short-read sequencing, we accurately defined the 5'- and 3'-ends of transcripts that are both poly(A) tailed and 5'-capped in glucose starvation, followed by proteome analysis. Our results confirm previous experimentally validated loci with alternative isoforms and reveal several transcriptome-wide patterns. First, we show that sense-antisense gene pairs are more strongly anticorrelated when a time lag is taken into account. Second, we show that the glucose starvation response initially elicits a shortening of 3'-UTRs and poly(A) tails, followed by a shortening of the 5'-UTRs at later time points. These result in domain gains and losses in proteins involved in the stress response. Finally, the relatively poor overlap both between differentially expressed genes (DEGs), differential transcript usage events (DTUs), and differentially detected proteins (DDPs) highlight the need for further study on post-transcriptional regulation mechanisms in glucose starvation.

Composition of Minor Ampullate Silk Makes Its Properties Different from Those of Major Ampullate Silk.

Spider's minor ampullate silk, or MI-silk, exhibits distinct mechanical properties and water resistance compared to its major ampullate counterpart (MA-silk). The principal protein constituent of MI-silk is known as minor ampullate spidroin, or MiSp, and while its sequence has been deciphered and is thought to underlie the differences in properties with MA-silk, the composition of MI-silk and the relationship between its composition and properties remain elusive. In this study, we set out to investigate the mechanical properties, water resistance, and proteome of MA-silk and MI-silk from Araneus ventricosus and Trichonephila clavata. We also synthesized artificial fibers from major ampullate spidroin, MaSp1 and 2, and MiSp to compare their properties. Our proteomic analysis reveals that the MI-silk of both araneids is composed of MiSp, MaSp1, and spidroin constituting elements (SpiCEs). The absence of MaSp2 in the MI-silk proteome and the comparison of the water resistance of artificial fibers suggest that the presence of MaSp2 is the reason for the disparity in water resistance between MI-silk and MA-silk.

Temporal phosphoproteomic analysis of VEGF-A signaling in HUVECs: an insight into early signaling events associated with angiogenesis.

Vascular endothelial growth factor-A (VEGF-A) is one of the primary factors promoting angiogenesis in endothelial cells. Although defects in VEGF-A signaling are linked to diverse pathophysiological conditions, the early phosphorylation-dependent signaling events pertinent to VEGF-A signaling remain poorly defined. Hence, a temporal quantitative phosphoproteomic analysis was performed in human umbilical vein endothelial cells (HUVECs) treated with VEGF-A-165 for 1, 5 and 10 min. This led to the identification and quantification of 1971 unique phosphopeptides corresponding to 961 phosphoproteins and 2771 phosphorylation sites in total. Specifically, 69, 153, and 133 phosphopeptides corresponding to 62, 125, and 110 phosphoproteins respectively, were temporally phosphorylated at 1, 5, and 10 min upon addition of VEGF-A. These phosphopeptides included 14 kinases, among others. This study also captured the phosphosignaling events directed through RAC, FAK, PI3K-AKT-MTOR, ERK, and P38 MAPK modules with reference to our previously assembled VEGF-A/VEGFR2 signaling pathway map in HUVECs. Apart from a significant enrichment of biological processes such as cytoskeleton organization and actin filament binding, our results also suggest a role of AAK1-AP2M1 in the regulation of VEGFR endocytosis. Taken together, the temporal quantitative phosphoproteomics analysis of VEGF signaling in HUVECs revealed early signaling events and we believe that this analysis will serve as a starting point for the analysis of differential signaling across VEGF members toward the full elucidation of their role in the angiogenesis processes. Workflow for the identification of early phosphorylation events induced by VEGF-A-165 in HUVEC cells.

Preliminary study of the safety of acrinol in probe-based confocal laser endomicroscopy during transurethral resection of bladder tumors.

We previously reported that probe-based confocal laser endomicroscopy using acrinol can depict cancerous nuclei. The objective of this study was to confirm the safety of acrinol in patients. For all seven patients, '50 ml' of a 0.1% acrinol and '1 ml' of 10% fluorescein in 99 ml of normal saline were introduced into the bladder. The laser probe adhered to the suspicious lesion from the working channel of the cystoscope. The patients underwent mucosal biopsy and transurethral resection after observation. Adverse events were noted during a valuation using common terminology criteria for adverse events version 4.0. Confocal laser endomicroscopy detected the nuclei of cancer cells in all seven patients. No adverse event was observed in any of the seven patients. Confocal laser endomicroscopy using acrinol as a novel dye can help visualize the cancerous nuclei of bladder urothelial carcinoma during cystoscopy without severe adverse events.

Shotgun proteomic investigation of methyltransferase and methylation profiles in lipopolysaccharide stimulated RAW264.7 murine macrophages.

Arginine methylation is a common post-translational modification which functions as an epigenetic regulator of transcription and plays a key role in various cell signaling pathways. The methylation of arginine residues is catalyzed by protein arginine methyltransferase (PRMT). However, the expression pattern and underlying mechanism of PRMTs and protein methylation profile in lipopolysaccharide (LPS)-induced innate immune responses are poorly understood. Using a shotgun proteomic approach, we found that LPS stimulation increased arginine and proline metabolism and responses to inflammation and bacterial infections. In comparison, cysteine and methionine metabolism, the pentose phosphate pathway, purine metabolism, and protein methylation factors were also decreased in LPS stimulated murine macrophage cell lines. We revealed that LPS stimulation downregulated PRMT1, PRMT5, and protein arginine methylation profiles in RAW264.7 cells using western blot analysis. Additionally, this phenomenon occurred in parallel with nitric oxide accumulation in LPS-induced macrophages. Using inflammation models, we demonstrate for the first time that LPS stimulation decreases PRMTs, leading to the decreasing of arginine methylation in macrophages.

Comprehensive Transcriptome Sequencing of Tanaidacea with Proteomic Evidences for Their Silk.

Tanaidaceans are small benthic crustaceans that mainly inhabit diverse marine environments, and they comprise one of the most diverse and abundant macrofaunal groups in the deep sea. Tanaidacea is one of the most thread-dependent taxa in the Crustacea, constructing tubes, spun with their silk, for shelter. In this work, we sequenced and assembled the comprehensive transcriptome of 23 tanaidaceans encompassing 14 families and 4 superfamilies of Tanaidacea, and performed silk proteomics of Zeuxo ezoensis to search for its silk genes. As a result, we identified two families of silk proteins that are conserved across the four superfamilies. The long and repetitive nature of these silk genes resembles that of other silk-producing organisms, and the two families of proteins are similar in composition to silkworm and caddisworm fibroins, respectively. Moreover, the amino acid composition of the repetitive motifs of tanaidacean silk tends to be more hydrophilic, and therefore could be a useful resource in studying their unique adaptation of silk use in a marine environment. The availability of comprehensive transcriptome data in these taxa, coupled with proteomic evidence of their silk genes, will facilitate evolutionary and ecological studies.

Darwin's bark spider shares a spidroin repertoire with Caerostris extrusa but achieves extraordinary silk toughness through gene expression.

Spider silk is a protein-based material whose toughness suggests possible novel applications. A particularly fascinating example of silk toughness is provided by Darwin's bark spider (Caerostris darwini) found in Madagascar. This spider produces extraordinarily tough silk, with an average toughness of 350 MJ m-1 and over 50% extensibility, and can build river-bridging webs with a size of 2.8 m2. Recent studies have suggested that specific spidroins expressed in C. darwini are responsible for the mechanical properties of its silk. Therefore, a more comprehensive investigation of spidroin sequences, silk thread protein contents and phylogenetic conservation among closely related species is required. Here, we conducted genomic, transcriptomic and proteomic analyses of C. darwini and its close relative Caerostris extrusa. A variety of spidroins and low-molecular-weight proteins were found in the dragline silk of these species; all of the genes encoding these proteins were conserved in both genomes, but their genes were more expressed in C. darwini. The potential to produce very tough silk is common in the genus Caerostris, and our results may suggest the existence of plasticity allowing silk mechanical properties to be changed by optimizing related gene expression in response to the environment.

Multicomponent nature underlies the extraordinary mechanical properties of spider dragline silk.

Dragline silk of golden orb-weaver spiders (Nephilinae) is noted for its unsurpassed toughness, combining extraordinary extensibility and tensile strength, suggesting industrial application as a sustainable biopolymer material. To pinpoint the molecular composition of dragline silk and the roles of its constituents in achieving its mechanical properties, we report a multiomics approach, combining high-quality genome sequencing and assembly, silk gland transcriptomics, and dragline silk proteomics of four Nephilinae spiders. We observed the consistent presence of the MaSp3B spidroin unique to this subfamily as well as several nonspidroin SpiCE proteins. Artificial synthesis and the combination of these components in vitro showed that the multicomponent nature of dragline silk, including MaSp3B and SpiCE, along with MaSp1 and MaSp2, is essential to realize the mechanical properties of spider dragline silk.

Metabolomic Analysis of Small Extracellular Vesicles Derived from Pancreatic Cancer Cells Cultured under Normoxia and Hypoxia.

Extracellular vesicles (EVs) released from cancer cells contribute to various malignant phenotypes of cancer, including metastasis, cachexia, and angiogenesis. Although DNA, mRNAs, miRNAs, and proteins contained in EVs have been extensively studied, the function of metabolites in EVs remains unclear. In this study, we performed a comprehensive metabolomic analysis of pancreatic cancer cells, PANC-1, cultured under different oxygen concentrations, and small EVs (sEVs) released from them, considering the fact that hypoxia contributes to the malignant behavior of cells in pancreatic cancer, which is a poorly diagnosed cancer. sEVs were collected by ultracentrifugation, and hydrophilic metabolites were analyzed using capillary ion chromatography-mass spectrometry and liquid chromatography-mass spectrometry, and lipids were analyzed by supercritical fluid chromatography-tandem mass spectrometry. A total of 140 hydrophilic metabolites and 494 lipids were detected in sEVs, and their profiles were different from those in cells. In addition, the metabolomic profile of sEVs was observed to change under hypoxic stress, and an increase in metabolites involved in angiogenesis was also detected. We reveal the hallmark of the metabolites contained in sEVs and the effect of tumor hypoxia on their profiles, which may help in understanding EV-mediated cancer malignancy.

Proteomic evidence for the silk fibroin genes of spider mites (Order Trombidiformes: Family Tetranychidae).

Spider mites are a group of arachnids belonging to Acari (mites and ticks), family Tetranychidae, known to produce nanoscale silk fibers characterized by a high Young's modulus. The silk fibroin gene of spider mites has been computationally predicted through genomic analysis of Tetranychus urticae Koch, but it has yet to be confirmed by proteomic evidence. In this work, we sequenced and assembled the transcriptome from two genera of spider mites, Tetranychus kanzawai Kishida and Panonychus citri (McGregor), and combined it with silk proteomics of T. urticae and P. citri to characterize the fibroin genes through comparative genomics and multiomics analysis. As a result, two fibroins were identified, which were different genes than those previously predicted by computational methods. The amino acid composition and secondary structure suggest similarity to aciniform or cylindrical spidroins of spider silk, which partly mirrors their mechanical properties, exhibiting a high Young's modulus. The availability of full-length fibroin sequences of spider mites facilitates the study of the evolution of silk genes that sometimes emerge in multiple lineages in a convergent manner and in the industrial application of artificial protein fibers through the study of the amino acid sequence and the resulting mechanical properties of these silks. SIGNIFICANCE: Here we sequenced and assembled the transcriptome from two genera of spider mites, T. kanzawai and P. citri, and combined it with silk proteomics of T. urticae and P. citri to characterize the fibroin genes through comparative genomics and multiomics analysis. Spider mite silk is especially characterized by its extremely fine nano-scale diameter and high Young's modulus, even exceeding those of spider silks. The availability of full-length fibroin sequences of spider mites facilitates the study of the evolution of silk genes, which independently evolved in mites, insects, and spiders but yet show sequence convergence, and in the industrial application of artificial protein fibers through the study of the amino acid sequence and the resulting mechanical properties of these silks.

Reprogramming of glutamine metabolism via glutamine synthetase silencing induces cisplatin resistance in A2780 ovarian cancer cells.

BACKGROUND: Cisplatin (CDDP) significantly prolongs survival in various cancers, but many patients also develop resistance that results in treatment failure. Thus, this study aimed to elucidate the underlying mechanisms by which ovarian cancer cells acquire CDDP resistance. METHODS: We evaluated the metabolic profiles in CDDP-sensitive ovarian cancer A2780 cells and CDDP-resistant A2780cis cells using capillary electrophoresis-time-of-flight mass spectrometry (CE-TOFMS). We further examined the expression of glutamine metabolism enzymes using real-time PCR and Western blot analyses. Cell viability was accessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. RESULTS: The results showed that levels of glutamine, glutamate, and glutathione (GSH), a key drug resistance mediator synthesized from glutamate, were significantly elevated in A2780cis cells than those in A2780 cells. Furthermore, glutamine starvation decreased the GSH levels and CDDP resistance in A2780cis cells. Interestingly, the expression of glutamine synthetase (GS/GLUL), which synthesizes glutamine from glutamate and thereby negatively regulates GSH production, was almost completely suppressed in resistant A2780cis cells. In addition, treatment of A2780cis cells with 5-aza-2'-deoxycytidine, a DNA-demethylating agent, restored GS expression and reduced CDDP resistance. In contrast, GS knockdown in CDDP-sensitive A2780 cells induced CDDP resistance. CONCLUSIONS: The results indicate that upregulation of GSH synthesis from glutamine via DNA methylation-mediated silencing of GS causes CDDP resistance in A2780cis cells. Therefore, glutamine metabolism could be a novel therapeutic target against CDDP resistance.

Spidroin profiling of cribellate spiders provides insight into the evolution of spider prey capture strategies.

Orb-weaving spiders have two main methods of prey capture: cribellate spiders use dry, sticky capture threads, and ecribellate spiders use viscid glue droplets. Predation behaviour is a major evolutionary driving force, and it is important on spider phylogeny whether the cribellate and ecribellate spiders each evolved the orb architecture independently or both strategies were derived from an ancient orb web. These hypotheses have been discussed based on behavioural and morphological characteristics, with little discussion on this subject from the perspective of molecular materials of orb web, since there is little information about cribellate spider-associated spidroin genes. Here, we present in detail a spidroin catalogue of six uloborid species of cribellate orb-weaving spiders, including cribellate and pseudoflagelliform spidroins, with transcriptome assembly complemented with long read sequencing, where silk composition is confirmed by proteomics. Comparative analysis across families (Araneidae and Uloboridae) shows that the gene architecture, repetitive domains, and amino acid frequencies of the orb web constituting silk proteins are similar among orb-weaving spiders regardless of the prey capture strategy. Notably, the fact that there is a difference only in the prey capture thread proteins strongly supports the monophyletic origin of the orb web.

Pre-treatment with D942, a furancarboxylic acid derivative, increases desiccation tolerance in an anhydrobiotic tardigrade Hypsibius exemplaris.

The tardigrade Hypsibius exemplaris can undergo anhydrobiosis. Several chemicals that inhibit successful anhydrobiosis in H. exemplaris have been identified, and these chemicals inhibit the activity of signaling molecules. In the present study, we investigated whether upregulation of the activity of these signaling molecules could improve desiccation tolerance of H. exemplaris. Pre-treatment with an indirect activator of AMP-activated protein kinase [AMPK; which directly inhibits mammalian NAD(P)H dehydrogenase [quinone] 1 [NQO1] of mitochondrial complex I (D942)] significantly improved desiccation tolerance of H. exemplaris, whereas a direct activator of AMPK did not. To elucidate the underlying molecular mechanisms, we examined the proteome of tardigrades treated with D942. Two proteins, putative glutathione S-transferase and pirin-like protein, were upregulated by treatment. Both of these proteins are known to be associated with the response to oxidative stress. One of the downregulated proteins was serine/threonine-proteinphosphatase 2A (PP2A) 65-kDa regulatory subunit A alpha isoform, and it is interesting to note that PP2A activity was previously suggested to be required for successful anhydrobiosis in H. exemplaris. Taken together, our results suggest that D942 treatment may partially induce responses common to those of desiccation stress. The identification of a chemical that improves desiccation tolerance of H. exemplaris may facilitate further investigation into desiccation tolerance mechanisms.

Proteomic and metabolomic analyses uncover sex-specific regulatory pathways in mouse fetal germline differentiation†.

Regulatory mechanisms of germline differentiation have generally been explained via the function of signaling pathways, transcription factors, and epigenetic regulation; however, little is known regarding proteomic and metabolomic regulation and their contribution to germ cell development. Here, we conducted integrated proteomic and metabolomic analyses of fetal germ cells in mice on embryonic day (E)13.5 and E18.5 and demonstrate sex- and developmental stage-dependent changes in these processes. In male germ cells, RNA processing, translation, oxidative phosphorylation, and nucleotide synthesis are dominant in E13.5 and then decline until E18.5, which corresponds to the prolonged cell division and more enhanced hyper-transcription/translation in male primordial germ cells and their subsequent repression. Tricarboxylic acid cycle and one-carbon pathway are consistently upregulated in fetal male germ cells, suggesting their involvement in epigenetic changes preceding in males. Increased protein stability and oxidative phosphorylation during female germ cell differentiation suggests an upregulation of aerobic energy metabolism, which likely contributes to the proteostasis required for oocyte maturation in subsequent stages. The features elucidated in this study shed light on the unrevealed mechanisms of germ cell development.

AMPK activity is required for the induction of anhydrobiosis in a tardigrade Hypsibius exemplaris, and its potential up-regulator is PP2A.

The anhydrobiotic tardigrade, Hypsibius exemplaris, was previously considered to require de novo gene expression and protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) activity for successful anhydrobiosis. These indicate that H. exemplaris has signal transduction systems responding to desiccation stress, with the involvement of phosphorylation events. To this end, we carried out time-series phosphoproteomics of H. exemplaris exposed to mild desiccation stress and detected 48 phosphoproteins with significant differential regulations. Among them, immediate and successive reduction of phosphorylation levels of AMP-activated protein kinase (AMPK) was observed. The subsequent chemical genetic approach showed that AMPK was activated during the preconditioning stage for anhydrobiosis, and inhibition of its activity impaired successful anhydrobiosis. As PP2A is known to dephosphorylate AMPK in other organisms, we suggested that decreased phosphorylation levels of AMPK upon mild desiccation stress were caused by dephosphorylation by PP2A. Accordingly, phosphoproteomics of animals pre-treated with the PP1/PP2A inhibitor cantharidic acid (CA) lacked the decrease in phosphorylation levels of AMPK. These observations suggest that AMPK activity is required for successful anhydrobiosis in H. exemplaris, and its phosphorylation state is possibly regulated by PP2A.

IMP dehydrogenase-2 drives aberrant nucleolar activity and promotes tumorigenesis in glioblastoma.

In many cancers, high proliferation rates correlate with elevation of rRNA and tRNA levels, and nucleolar hypertrophy. However, the underlying mechanisms linking increased nucleolar transcription and tumorigenesis are only minimally understood. Here we show that IMP dehydrogenase-2 (IMPDH2), the rate-limiting enzyme for de novo guanine nucleotide biosynthesis, is overexpressed in the highly lethal brain cancer glioblastoma. This leads to increased rRNA and tRNA synthesis, stabilization of the nucleolar GTP-binding protein nucleostemin, and enlarged, malformed nucleoli. Pharmacological or genetic inactivation of IMPDH2 in glioblastoma reverses these effects and inhibits cell proliferation, whereas untransformed glia cells are unaffected by similar IMPDH2 perturbations. Impairment of IMPDH2 activity triggers nucleolar stress and growth arrest of glioblastoma cells even in the absence of functional p53. Our results reveal that upregulation of IMPDH2 is a prerequisite for the occurance of aberrant nucleolar function and increased anabolic processes in glioblastoma, which constitutes a primary event in gliomagenesis.

Orb-weaving spider Araneus ventricosus genome elucidates the spidroin gene catalogue.

Members of the family Araneidae are common orb-weaving spiders, and they produce several types of silks throughout their behaviors and lives, from reproduction to foraging. Egg sac, prey capture thread, or dragline silk possesses characteristic mechanical properties, and its variability makes it a highly attractive material for ecological, evolutional, and industrial fields. However, the complete set of constituents of silks produced by a single species is still unclear, and novel spidroin genes as well as other proteins are still being found. Here, we present the first genome in genus Araneus together with the full set of spidroin genes with unamplified long reads and confirmed with transcriptome of the silk glands and proteome analysis of the dragline silk. The catalogue includes the first full length sequence of a paralog of major ampullate spidroin MaSp3, and several spider silk-constituting elements designated SpiCE. Family-wide phylogenomic analysis of Araneidae suggests the relatively recent acquisition of these genes, and multiple-omics analyses demonstrate that these proteins are critical components in the abdominal spidroin gland and dragline silk, contributing to the outstanding mechanical properties of silk in this group of species.

Comparative proteome analysis of the capsule from patients with frozen shoulder.

BACKGROUND: The etiology of frozen shoulder (FS) is unclear. Accordingly, this study used a label-free quantitative shotgun proteomic approach to elucidate the pathogenesis of FS based on protein expression levels. METHODS: Tissue samples from the rotator interval (RI), middle glenohumeral ligament (MGHL), and anterior-inferior glenohumeral ligament (IGHL) were collected from 12 FSs with severe stiffness and 7 shoulders with a rotator cuff tear (RCT) as controls. Protein mixtures were digested and analyzed by nano-liquid chromatography/electrospray ionization-tandem mass spectrometry. Relative protein expression levels were calculated by the signal intensity of identified peptide ions on mass spectra. Differentially expressed proteins between FS and RCT samples were evaluated by a gene enrichment analysis using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes. RESULTS: We identified 1594 proteins, 1358 of which were expressed in all 6 tissue groups. We detected more upregulated proteins in the upper (RI and MGHL) FS groups and the lower (IGHL) RCT group than in the comparative groups, respectively. Various proteins with functions in tissue repair, collagen metabolism and fibrillation, cell-cell and cell-matrix adhesion, blood coagulation, and the immune response were expressed more highly in the RI and MGHL FS groups than in the RCT group. Proteins with functions in phagocytosis, glutathione metabolism, retinoid metabolism, and cholesterol metabolism were expressed more highly in the IGHL RCT group than in the FS group. CONCLUSIONS: The pathophysiology of FS differs between the upper and lower parts of the joint capsule. Different treatment strategies for FS may be appropriate, depending on the location.

Probe-Based Confocal Laser Endomicroscopy Using Acrinol as a Novel Dye Can Be Used to Observe Cancer Nuclei of Bladder Carcinoma In Situ.

Background: Cystoscopy using white light is a standard procedure for diagnosing bladder cancer; however, white light can result in missed lesions that are present, but not visible, such as in cases of carcinoma in situ (CIS). In this case report, we describe observing the nuclei of urothelial carcinoma cells in situ that were not visible with cystoscopy under white light using probe-based confocal laser endomicroscopy (pCLE) with acrinol and fluorescein during transurethral resection of a bladder tumor (TURBT). Case Presentation: A 59-year-old male with a medical history of neurogenic bladder dysfunction with multiple bladder diverticula was referred to the urology department for gross hematuria. TURBT was performed with the assistance of pCLE, using acrinol as a novel dye. Standard cystoscopy under white light could not detect any bladder tumor; however, pCLE using acrinol could detect the abnormal nuclei of bladder CIS. Subsequent histopathologic analysis of the specimen confirmed a diagnosis of bladder CIS. To our knowledge, this is the first reported case of bladder CIS diagnosed with the assistance of pCLE using acrinol in a patient undergoing a TURBT. Conclusion: pCLE using acrinol as a novel dye can help observe the cancerous nuclei of bladder CIS that cannot be detected using conventional cystoscopy under white light. Therefore, pCLE using acrinol is one possible modality for performing an optical biopsy during TURBT.

Systematic characterization of artificial small RNA-mediated inhibition of Escherichia coli growth.

A new screening system for artificial small RNAs (sRNAs) that inhibit the growth of Escherichia coli was constructed. In this system, we used a plasmid library to express RNAs of ∼120 nucleotides, each with a random 30-nucleotide sequence that can recognize its target mRNA(s). After approximately 60,000 independent colonies were screened, several plasmids that inhibited bacterial growth were isolated. To understand the inhibitory mechanism, we focused on one sRNA, S-20, that exerted a strong inhibitory effect. A time-course analysis of the proteome of S-20-expressing E. coli and a bioinformatic analysis were used to identify potential S-20 target mRNAs, and suggested that S-20 binds the translation initiation sites of several mRNAs encoding enzymes such as peroxiredoxin (osmC), glycyl-tRNA synthetase α subunit (glyQ), uncharacterized protein ygiM, and tryptophan synthase ß chain (trpB). An in vitro translation analysis of chimeric luciferase-encoding mRNAs, each containing a potential S-20 target sequence, indicated that the translation of these mRNAs was inhibited in the presence of S-20. A gel shift analysis combined with the analysis of a series of S-20 mutants suggested that S-20 targets multiple mRNAs that are responsible for inhibiting E. coli growth. These data also suggest that S-20 acts like an endogenous sRNA and that E. coli can utilize artificial sRNAs.