TRUE Gene Silencing.

TRUE gene silencing is an RNA-mediated gene expression control technology and is termed after tRNase ZL-utilizing efficacious gene silencing. In this review, I overview the potentiality of small guide RNA (sgRNA) for TRUE gene silencing as novel therapeutics. First, I describe the physiology of tRNase ZL and cellular small RNA, and then sgRNA and TRUE gene silencing. An endoribonuclease, tRNase ZL, which can efficiently remove a 3' trailer from pre-tRNA, is thought to play the role in tRNA maturation in the nucleus and mitochondria. There exist various small RNAs including miRNA and fragments from tRNA and rRNA, which can function as sgRNA, in living cells, and human cells appear to be harnessing cytosolic tRNase ZL for gene regulation together with these small RNAs. By utilizing the property of tRNase ZL to recognize and cleave micro-pre-tRNA, a pre-tRNA-like or micro-pre-tRNA-like complex, as well as pre-tRNA, tRNase ZL can be made to cleave any target RNA at any desired site under the direction of an artificial sgRNA that binds a target RNA and forms the pre-tRNA-like or micro-pre-tRNA-like complex. This general RNA cleavage method underlies TRUE gene silencing. Various examples of the application of TRUE gene silencing are reviewed including the application to several human cancer cells in order to induce apoptosis. Lastly, I discuss the potentiality of sgRNA as novel therapeutics for multiple myeloma.

Genes for tRNA recycling are upregulated in response to infection with Theiler's mouse encephalitis virus.

The concept of tRNA recycling has recently emerged from the studies of ribosome-associated quality control. Therein tRNase ZS removes the 2', 3'>p from the ANKZF1-cleaved tRNA and the subsequent TRNT1 action re-generates the intact tRNA. To know the roles of the tRNA recycling in vivo, we investigated how viral infection affects the tRNA recycling system by analyzing the mRNA levels of tRNase ZS and TRNT1. We found that both genes in HeLa cells are upregulated in response to infection of Theiler's mouse encephalitis virus but not to that of an influenza A virus. Upregulation was also observed in cells infected with encephalomyocarditis virus with reduced efficiency. The levels of the IFN-ß mRNA appeared to positively correlate with those of the tRNase ZS and TRNT1 mRNAs. The tRNase ZS gene may be regulated post-transcriptionally in the cells infected with Theiler's mouse encephalitis virus.

Perturbing the Normal Level of SIDT1 Suppresses the Naked ASO Effect.

Although antisense oligonucleotide (ASO) therapeutics can be taken up by living cells without carrier molecules, a large part of incorporated ASOs are trapped in the endosomes and do not exert therapeutic effects. To improve their therapeutic effects, it would be important to elucidate the mechanism of cellular uptake and intracellular trafficking of ASOs. In this study, we investigated how SIDT1 affects cellular uptake and intracellular trafficking of ASOs. Fluorescence microscopic analysis suggested that most of naked ASOs are trafficked to the lysosomes via the endosomes. The data obtained from flow cytometry and fluorescence microscopy together showed that although the SIDT1 level barely affects the total cellular uptake of ASOs, it appears to affect the intracellular trafficking of ASOs. We also showed that SIDT1 exists mainly in the endoplasmic reticulum and that perturbing the normal level of SIDT1 suppresses the antisense effect of the naked ASO targeting miR-16.

Transcription from the proximal promoter of ELAC1, a gene for tRNA repair, is upregulated by interferons.

tRNase ZS (ELAC1) and TRNT1 function in tRNA recycling. Recently, we have shown that these genes are upregulated in the cells infected with Theiler's mouse encephalitis virus (TMEV), implying that tRNA recycling functions in response to viral infection. To address the molecular mechanism underlying the ELAC1 upregulation in the cells infected with TMEV, we performed luciferase assays using various plasmid constructs harboring the ELAC1 promoter region. The luciferase expression from a construct containing the full-length ELAC1 promoter was augmented by TMEV, poly IC, IFN-ß, or IFN-γ. We identified four IFN-stimulated responsible elements (ISREs) in the proximal promoter region. The luciferase expression from the constructs that lack all the ISREs was strongly reduced compared with that from the constructs with the four ISREs in the presence of IFN-ß or IFN-γ. The observation that the ISREs from the ELAC1 promoter are essential for the gene upregulation by IFN-ß or IFN-γ suggests that the ELAC1 gene is upregulated by IFNs.

Suppression of Tumor Growth in a Rabbit Hepatic Cancer Model by Boron Neutron Capture Therapy With Liposomal Boron Delivery Systems.

BACKGROUND/AIM: Tumor cell destruction by boron neutron capture therapy (BNCT) is attributed to the nuclear reaction between 10B and thermal neutrons. The accumulation of 10B atoms in tumor cells without affecting adjacent healthy cells is crucial for effective BNCT. We previously reported that several types of liposomal boron delivery systems (BDS) delivered effective numbers of boron atoms to cancer tissues, and showed tumor-growth suppression after thermal neutron irradiation. In the present study, we examined the effects of BNCT after intra-arterial infusion of 10B-borono-dodecaborate (10BSH) by liposomal BDS in rabbit hepatic cancer models. MATERIALS AND METHODS: We prepared 10BSH-entrapped transferrin-conjugated polyethylene glycol liposomes constructed with distearoyl-boron lipid (TF-PEG-DSBL), and performed thermal neutron irradiation at the Kyoto University Institute for Integrated Radiation and Nuclear Science after intra-arterial infusion into rabbit VX-2 hepatic tumors. RESULTS: Concentrations of 10B in VX-2 tumors on delivery with TF-PEG-DSBL liposomes reached 25 ppm on day 3 after the injection. Tumor growth was suppressed by thermal neutron irradiation after intra-arterial injection of this 10BSH-containing liposomal BDS, without damage to normal cells. CONCLUSION: The present results demonstrate the applicability of 10B-containing TF-PEG-DSBL liposomes as a novel intra-arterial boron carrier in BNCT for cancer.

A naked antisense oligonucleotide with phosphorothioate linkages is taken up intracellularly more efficiently but functions less effectively.

We have been developing a gene silencing technology by harnessing a tRNA 3' processing endoribonuclease, tRNase ZL, with antisense oligonucleotides. Here, to further improve this technology, we investigated how the length and the modifications of naked oligonucleotides affect the efficiency of their uptake by HeLa, HEK293, and HL60 cells by flow cytometry and fluorescence microscopy. 7-30-nt Alexa-Fluor-568-labeled DNAs with phosphorothioate linkages and 7-30-nt Alexa-Fluor-568-labeled, 2'-O-methylated RNAs without phosphorothioate linkages were examined, and, on the whole, longer oligonucleotides were shown to be intracellularly taken up more efficiently. In addition, a 2'-O-methoxyethylated RNA without phosphorothioate linkages, a 2'-fluoriated RNA without phosphorothioate linkages, a 2'-O-methylated RNA with phosphorothioate linkages, and a 2'-O-methylated RNA with phosphorothioate linkages and LNA modifications of 5'-/3'-terminal nucleotides were examined. The oligonucleotides with phosphorothioate linkages were taken up by the cells more efficiently than those without the linkages. Furthermore, we examined how the phosphorothioate linkages of oligonucleotides affect their antisense effects using 22-nt anti-miR16 oligonucleotides with and without phosphorothioate linkages. The latter oligonucleotide decreased the miR16 level much more intensively than the former, although the latter was intracellularly taken up much less efficiently. These observations may be not generalized and differ depending on features of oligonucleotides and cell types. Taken together these results suggest that the productive uptake efficiency for an antisense oligonucleotide needs to be considered to select its length and modifications.

The heptamer sgRNA targeting the human OCT4 mRNA can upregulate the OCT4 expression.

TRUE gene silencing is one of the gene suppression technologies. This technology exploits the enzymatic property of the tRNA 3' processing endoribonuclease tRNase ZL, which is that it can cleave a target RNA under the direction of a small guide RNA (sgRNA). We have been working on the development of therapeutic sgRNAs for hematological malignancies. In the course of an experiment to examine the ability of the heptamer-type sgRNA H15792 targeting the OCT4 mRNA to differentiate human amnion stem cells, we observed unexpectedly that the amnion cells exhibited a morphology resembling initialized cells. Here we investigated the effect of H15792 on human HL60 leukemia cells, and found that H15792 can upregulate the OCT4 expression and the expression of alkaline phosphatase in the cells.

Heptamer-type small guide RNA that can shift macrophages toward the M1 state.

Multiple myeloma is a refractory cancer of plasma cells. Although treatment strategies for multiple myeloma are getting improved year by year, in most cases patients relapse due to the emergence of drug-resistant mutations in the myeloma cells. The interplay between myeloma cells and tumor-associated macrophages (TAM) is important for the pathology. We thought that some heptamer-type sgRNAs for TRUE gene silencing would be able to transform TAM toward the M1 state and might become therapeutic drugs for myeloma. Here, we searched for heptamer-type sgRNAs that can shift macrophages toward the M1 state. We screened a heptamer-type sgRNA library for the ability to up-regulate IL-12b gene expression in human macrophage-like cell lines, and found three such sgRNAs. One of the sgRNAs, H12960, which also showed such ability in human fresh macrophages and mouse macrophage-like cell lines, efficiently suppressed human myeloma cell growth in SCID/NOD mice.

RNA-hydrolyzing activity of metallo-ß-lactamase IMP-1.

Metallo-ß-lactamases (MBLs) hydrolyze a wide range of ß-lactam antibiotics. While all MBLs share a common αß/ßα-fold, there are many other proteins with the same folding pattern that exhibit different enzymatic activities. These enzymes, together with MBLs, form the MBL superfamily. Thermotoga maritima tRNase Z, a tRNA 3' processing endoribonuclease of MBL-superfamily, and IMP-1, a clinically isolated MBL, showed a striking similarity in tertiary structure, despite low sequence homology. IMP-1 hydrolyzed both total cellular RNA and synthetic small unstructured RNAs. IMP-1 also hydrolyzed pre-tRNA, but its cleavage site was different from those of T. maritima tRNase Z and human tRNase Z long form, indicating a key difference in substrate recognition. Single-turnover kinetic assays suggested that substrate-binding affinity of T. maritima tRNase Z is much higher than that of IMP-1.

Large-Scale Isolation of Three O-Methyl Anthocyanins from Bilberry (Vaccinium myrtillus L.) Extract.

Three O-methyl anthocyanidin 3-O-ß-D-glucopyranosides were isolated from bilberry extract on a large-scale basis together with two non O-methyl analogues. Anthocyanidin 3-O-ß-D-galactopyranosides were removed from bilberry extract together with parts of anthocyanidin 3-O-α-L-arabinopyranosides after treatment with ß-galactosidase. The remaining arabinopyranosides were removed by applying acid catalytic hydrolysis. The amounts of anthocyanins recovered as flavylium trifluoroacetic acid salt were as follows: 630 mg for petunidin 3-O-ß-D-glucopyranoside, 423 mg for peonidin 3-O-ß-D-glucopyranoside, 588 mg for malvidin 3-O-ß-D-glucopyranoside, 877 mg for delphinidin 3-O-ß-D-glucopyranoside, and 742 mg for cyanidin 3-O-ß-D-glucopyranoside.

Mastication Affects Transcriptomes of Mouse Microglia.

BACKGROUND/AIM: We investigated whether mastication affects microglia, whose activity is thought to be associated with cognition and brain tumor progression. MATERIALS AND METHODS: We kept mice by feeding either a hard or soft diet for 2, 4 or 8 months. After each period, we removed the whole brains and isolated microglia. The total RNA extracted from each brain's microglia was subjected to DNA microarray analysis. RESULTS: Many genes were found to be significantly differentially expressed between hard- and soft-diet-fed mice in each group of the same feeding period. The expression of several genes involved in the regulation of actin cytoskeleton was down-regulated in the soft-diet-fed mice. CONCLUSION: Mastication may affect microglia's roles in cognition as well as their neuroimmune activity through their activity of patrolling the brain.

The 31-nucleotide Y4-RNA fragment in plasma is a potential novel biomarker.

The 31- and 32-nt 5'-fragment of Y4-RNA (Y4RNAfr) exists abundantly in human peripheral blood plasma. Although physiological roles of the plasma Y4RNAfr are not well established, its potential utility as a diagnostic/prognostic marker for acute coronary syndrome was suggested. In this paper, to establish a normal range of the Y4RNAfr level in plasma, we measured plasma Y4RNAfr levels of 40 healthy persons using the method we have developed, and compared them with other blood test data. From the obtained data, we tentatively regarded <0.1 fmol/ng as normal for the Y4RNAfr level in peripheral blood plasma. And the white blood cell count (WBC) and the C-reactive protein (CRP) level showed moderate positive correlations with the Y4RNAfr level, suggesting that Y4RNAfr could be a potential novel inflammatory marker. We also measured the Y4RNAfr level in peripheral blood plasma from four multiple myeloma patients. The plasma Y4RNAfr level was abnormal in all four myeloma patients, and the levels for two patients were far beyond the normal level. The WBC for each patient was normal and the CRP levels for two patients were normal. These observations together suggest that a high level of Y4RNAfr in peripheral blood plasma and a normal WBC could be indicative of multiple myeloma.

Dual activity of PNGM-1 pinpoints the evolutionary origin of subclass B3 metallo-ß-lactamases: a molecular and evolutionary study.

Resistance to ß-lactams is one of the most serious problems associated with Gram-negative infections. ß-Lactamases are able to hydrolyze ß-lactams such as cephalosporins and/or carbapenems. Evolutionary origin of metallo-ß-lactamases (MBLs), conferring critical antibiotic resistance threats, remains unknown. We discovered PNGM-1, the novel subclass B3 MBL, in deep-sea sediments that predate the antibiotic era. Here, our phylogenetic analysis suggests that PNGM-1 yields insights into the evolutionary origin of subclass B3 MBLs. We reveal the structural similarities between tRNase Zs and PNGM-1, and demonstrate that PNGM-1 has both MBL and tRNase Z activities, suggesting that PNGM-1 is thought to have evolved from a tRNase Z. We also show kinetic and structural comparisons between PNGM-1 and other proteins including subclass B3 MBLs and tRNase Zs. These comparisons revealed that the B3 MBL activity of PNGM-1 is a promiscuous activity and subclass B3 MBLs are thought to have evolved through PNGM-1 activity.

Evaluation of double heptamer-type sgRNA as a potential therapeutic agent against multiple myeloma.

Emergence of drug-resistant mutations in the course of myeloma cell evolution and subsequent relapse of myeloma appears to be currently inevitable in most patients. To remedy this situation, we are trying to develop therapeutic small guide RNAs (sgRNAs) based on tRNase ZL-utilizing efficacious gene silencing (TRUE gene silencing), an RNA-mediated gene expression control technology. We designed two sets of double heptamer-type sgRNA, which target the human BCL2 mRNA. Both sets of double heptamer-type sgRNA reduced viability of human myeloma cell lines, RPMI-8226 and KMM-1. We also performed a mouse xenograft experiment to examine how the double heptamer-type sgRNA DHa1(BCL2)/DHa2(BCL2) can reduce the growth of KMM-1 cells in vivo. Median survival periods of the sgRNA cohorts were greater than that of the control cohort by 11-43 days. Furthermore, we designed two sets of double heptamer-type sgRNA, which target the human CCND1 mRNA, and both sets synergistically reduced RPMI-8226 cell viability.

Potential physiological roles of the 31/32-nucleotide Y4-RNA fragment in human plasma.

The 31- and 32-nt 5'-fragments of Y4-RNA (Y4RNAfr) exist abundantly in human plasma. The Y4RNAfr can function as 5'-half-tRNA-type sgRNA for tRNase ZL, although we do not know yet what its physiological roles are and what cellular RNAs are its genuine targets. In this paper, we analyzed the effects of the Y4RNAfr on cell viability and transcriptomes using HL60, RPMI-8226, and HEK293 cells, and Y4RNAfr-binding RNAs in A549 cells. Although the Y4RNAfr hardly affected the viability of HL60, RPMI-8226, and HEK293 cells, it significantly affected their transcriptome. The DAVID analysis for > 2-fold upregulated and downregulated genes suggested that the Y4RNAfr may affect various KEGG pathways. We obtained 108 Y4RNAfr-binding RNAs in A549 cells, searched potential secondary structures of complexes between theY4RNAfr and its binding RNAs for the pre-tRNA-like structure, and found many such structures. One of the five best fitted structures was for the MKI67 mRNA, suggesting that the Y4RNAfr can decrease the cellular MKI67 level through guiding the cleavage of the MKI67 mRNA by tRNase ZL. This may be one of the underlying mechanisms for the reported observation that the Y4RNAfr suppresses the proliferation of A549 cells.

Microbicidal Effects of Stored Aqueous Ozone Solution Generated by Nano-bubble Technology.

BACKGROUND/AIM: Clinically used disinfectants are often irritating and cause skin problems. Ozone water is unique among disinfectants. It does not damage skin cells and readily decomposes to oxygen without generating harmful residues. On the other hand, it rapidly loses its sanitizing activity. Recently developed nano-bubble ozone water (NBOW) can keep its sanitizing activity much longer. This study aimed to examine the microbicidal effects of NBOW after long-term storage. MATERIALS AND METHODS: The concentration of ozone in NBOW was examined by measuring the NBOW redox potential. Microbicidal activity was evaluated by colony formation assays, after incubating bacteria with NBOW for set time periods. RESULTS: NBOW lost its microbicidal activity after 1 year of storage at 4°C. Stocked frozen, NBOW retained appreciable microbicidal activity after 1 year of storage. Mycobacterium smegmatis, one of the most disinfectant-resistant bacteria, was killed within 15 min. NBOW was resistant to freeze-thawing. CONCLUSION: NBOW that had been stored frozen possessed sufficient microbicidal activity to kill bacteria even after 1 year of storage. Moreover, it was shown that NBOW is freeze-thaw resistant. NBOW possesses desirable features rendering it an attractive alternative disinfectant.

The Y4-RNA fragment, a potential diagnostic marker, exists in saliva.

The 94-nt full-length Y4-RNA is thought to have roles in the initiation of DNA replication and RNA quality control. Although its 31/32-nt fragment also exists abundantly in plasma, little is known about its physiological role. Since the 31/32-nt Y4-RNA fragment in sera is reported to be more abundant in patients with coronary artery disease than healthy persons, the fragment may have a potential for a diagnostic and/or prognostic biomarker for some diseases regardless of its functionality. As a step toward further investigation of its potential utility, we examined if the 31/32-nt Y4-RNA fragment also exists in saliva that can be obtained noninvasively, and showed that, in addition to the 31/32-nt fragment, 14- and 11-nt Y4-RNA fragments are present in all saliva RNA samples from four healthy persons. We established a PCR method to accurately quantitate the amount of the 31/32-nt Y4-RNA fragment, and estimated its amount in saliva of healthy persons to be 0.06 ± 0.04 fmol per nanogram of saliva RNA. We also tried to develop an easier quantitation method using a DNA molecular beacon.

TRUE Gene Silencing: Screening of a Heptamer-type Small Guide RNA Library for Potential Cancer Therapeutic Agents.

TRUE gene silencing (termed after tRNase Z(L)-utilizing efficacious gene silencing) is one of the RNA-directed gene silencing technologies, which utilizes an artificial small guide RNA (sgRNA) to guide tRNA 3' processing endoribonuclease, tRNase Z(L), to recognize a target RNA. sgRNAs can be taken up by cells without any transfection reagents and can downregulate their target RNA levels and/or induce apoptosis in human cancer cells. We have screened an sgRNA library containing 156 heptamer-type sgRNAs for the effect on viability of human myeloma and leukemia cells, and found that 20 of them can efficiently induce apoptosis in at least one of the cancer cell lines. Here we present a protocol for screening of a heptamer-type sgRNA library for potential therapeutic drugs against blood cancers. The protocol includes how to construct the sgRNA library, how to assess the effect of each sgRNA on cell viability, and how to further evaluate the effective sgRNAs by flow cytometry. Around 2,000 hits would be expected to be obtained by screening the full-scale sgRNA library composed of 16,384 heptamers.

Involvement of an intracellular vesicular transport process in naked-sgRNA-mediated TRUE gene silencing.

tRNase ZL-utilizing efficacious gene silencing (TRUE gene silencing) is an RNA-mediated gene expression control technology with therapeutic potential. Recently, our group demonstrated that a heptamer, mh1 (Bcl­2), targeting human Bcl-2 mRNA, can be taken up by cells without the use of any transfection reagents and can induce the apoptosis of leukemia cells. However, little is known regarding the mechanism of naked small guide (sg)RNA uptake by cultured cells. Therefore, in the present study the effects of various inhibitors on the induction of apoptosis by naked sgRNA treatment were investigated in order to identify the uptake pathway required for sgRNA function in cultured cells. Addition of the endocytosis inhibitors chlorpromazine, nystatin or methyl­ß­cyclodextrin together with naked effective sgRNA was unable to diminish the apoptosis­inducing effects of naked sgRNA or the reduction in target mRNA, suggesting that functional uptake of sgRNA by cells is clathrin­, caveolae­ and raft­independent. Next, chloroquine, an inhibitor of lysosome acidification, and brefeldin A, an inhibitor that blocks protein transport from the Golgi apparatus to the endoplasmic reticulum were administered. In the presence of these compounds, the apoptosis­inducing effects of naked sgRNA were reduced. These results suggest that a vesicular transport process is involved in sgRNA­mediated TRUE gene silencing. A greater understanding of how naked sgRNAs enter cells and how they reach their target RNAs may aid in the design of more specifically­targeted and potent sgRNA drugs.

Potential small guide RNAs for tRNase ZL from human plasma, peripheral blood mononuclear cells, and cultured cell lines.

Several pieces of evidence suggest that small RNA degradation products together with tRNase ZL appear to form another layer of the whole gene regulatory network. The degraded RNA such as a 5'-half-tRNA and an rRNA fragment function as small guide RNA (sgRNA) to guide the enzyme to target RNA. We were curious whether there exist RNAs in plasma that can function as sgRNAs for tRNase ZL, whether these RNAs are working as signaling molecules between cells to fulfill physiological roles, and whether there are any differences in plasma sgRNA species and levels between normal and pathological conditions. Here, we analyzed small plasma RNAs from three healthy persons and three multiple myeloma patients for potential sgRNAs by deep sequencing. We also examined small RNAs from peripheral blood mononuclear cells (PBMC) of three healthy persons and three myeloma patients and from various cultured human cell lines for sgRNAs. We found that read-number distribution patterns of plasma and PBMC RNAs differ between persons in the range of 5-40 nt and that there are many RNA species that exist significantly more or less abundantly in the plasma or PBMC of the myeloma patients than those of the healthy persons. Furthermore, we found that there are many potential sgRNAs in the 5-40-nt RNAs and that, among them, a 31-nt RNA fragment derived from 94-nt Y4-RNA, which can function as a 5'-half-tRNA-type sgRNA, is overwhelmingly abundant in the plasma of 2/3 of the examinees. These observations suggest that the gene regulatory network via tRNase ZL and sgRNA may be extended intercellularly.