Hidenori Tani

The RNA Degradation Pathway Regulates the Function of GAS5 a Non-Coding RNA in Mammalian Cells

Studies of various mRNAs have revealed that changes in the abundance of transcripts, through mRNA degradation, act as a critical step in the control of various biological pathways. Similarly, the regulation of non-coding RNA (ncRNA) levels is also considered to be important for their biological functions; however, far less is known about the mechanisms and biological importance of ncRNA turnover for the regulation of ncRNA functions. The growth arrest-specific 5 (GAS5) ncRNA accumulates during growth arrest induced by serum starvation and its transcript is degraded by the well characterized nonsense-mediated RNA decay (NMD) pathway. Historically, NMD was discovered as a RNA quality control system to eliminate aberrant transcripts; however, accumulating evidence shows that NMD also regulates the abundance of physiological transcripts. Interestingly, the GAS5 transcript has the ability to bind the glucocorticoid receptor (GR), resulting in the inhibition of its ligand-dependent association with DNA. The GR binds the promoters of various glucocorticoid-responsive genes, including apoptosis-related genes. In this study, we examined whether the RNA degradation pathway can regulate this function of GAS5. We measured the steady-state abundance and the decay rate of GAS5 in UPF1-depleted human cells using the 5′-bromo-uridine immunoprecipitation chase (BRIC) method, an inhibitor-free method for directly measuring RNA stability. We found that levels of the GAS5 transcript were elevated owing to prolonged decay rates in response to UPF1 depletion, and consequently the apoptosis-related genes, cIAP2 and SGK1, were down-regulated. In addition, serum starvation also increased the transcript levels of GAS5 because of prolonged decay rates, and conversely decreased levels of cIAP2 and SGK1 mRNA. Taken together, we found that the RNA degradation pathway can regulate the function of the GAS5 ncRNA in mammalian cells.

Identification of hundreds of novel UPF1 target transcripts by direct determination of whole transcriptome stability

UPF1 eliminates aberrant mRNAs harboring premature termination codons, and regulates the steady-state levels of normal physiological mRNAs. Although genome-wide studies of UPF1 targets performed, previous studies did not distinguish indirect UPF1 targets because they could not determine UPF1-dependent altered RNA stabilities. Here, we measured the decay rates of the whole transcriptome in UPF1-depleted HeLa cells using BRIC-seq, an inhibitor-free method for directly measuring RNA stability. We determined the half-lives and expression levels of 9,229 transcripts. An amount of 785 transcripts were stabilized in UPF1-depleted cells. Among these, the expression levels of 76 transcripts were increased, but those of the other 709 transcripts were not altered. RNA immunoprecipitation showed UPF1 bound to the stabilized transcripts, suggesting that UPF1 directly degrades the 709 transcripts. Many UPF1 targets in this study were newly identified. This study clearly demonstrates that direct determination of RNA stability is a powerful approach for identifying targets of RNA degradation factors.

Genome-wide technology for determining RNA stability in mammalian cells: Historical perspective and recent advantages based on modified nucleotide labeling

Changing the abundance of transcripts by regulated RNA degradation is a critical step in the control of various biological pathways. Recently, genome-wide inhibitor-free technologies for determining RNA stabilities in mammalian cells have been developed. In these methods, endogenous RNAs are pulse labeled by uridine analogs [e.g., 4-thiouridine (4sU), 5-etyniluridine (EU) and 5′-bromo-uridine (BrU)], followed by purification of labeled de novo RNAs. These technologies have revealed that the specific half-life of each mRNA is closely related to its physiological function. Genes with short-lived mRNAs are significantly enriched among regulatory genes, while genes with long-lived mRNAs are enriched among housekeeping genes. This review describes the recent progress of experimental procedures for measuring RNA stability.

Identification and Characterization of Novel Genotoxic Stress-Inducible Nuclear Long Noncoding RNAs in Mammalian Cells

Whole transcriptome analyses have revealed a large number of novel transcripts including long and short noncoding RNAs (ncRNAs). Currently, there is great interest in characterizing the functions of the different classes of ncRNAs and their relevance to cellular processes. In particular, nuclear long ncRNAs may be involved in controlling various aspects of biological regulation, such as stress responses. By a combination of bioinformatic and experimental approaches, we identified 25 novel nuclear long ncRNAs from 6,088,565 full-length human cDNA sequences. Some nuclear long ncRNAs were conserved among vertebrates, whereas others were found only among primates. Expression profiling of the nuclear long ncRNAs in human tissues revealed that most were expressed ubiquitously. A subset of the identified nuclear long ncRNAs was induced by the genotoxic agents mitomycin C or doxorubicin, in HeLa Tet-off cells. There were no commonly altered nuclear long ncRNAs between mitomycin C- and doxorubicin-treated cells. These results suggest that distinct sets of nuclear long ncRNAs play roles in cellular defense mechanisms against specific genotoxic agents, and that particular long ncRNAs have the potential to be surrogate indicators of a specific cell stress.

Inhibition of Hepatitis C Virus NS3 Helicase by Manoalide

The hepatitis C virus (HCV) causes one of the most prevalent chronic infectious diseases in the world, hepatitis C, which ultimately develops into liver cancer through cirrhosis. The NS3 protein of HCV possesses nucleoside triphosphatase (NTPase) and RNA helicase activities. As both activities are essential for viral replication, NS3 is proposed as an ideal target for antiviral drug development. In this study, we identified manoalide (1) from marine sponge extracts as an RNA helicase inhibitor using a high-throughput screening photoinduced electron transfer (PET) system that we previously developed. Compound 1 inhibits the RNA helicase and ATPase activities of NS3 in a dose-dependent manner, with IC(50) values of 15 and 70 μM, respectively. Biochemical kinetic analysis demonstrated that 1 does not affect the apparent K(m) value (0.31 mM) of NS3 ATPase activity, suggesting that 1 acts as a noncompetitive inhibitor. The binding of NS3 to single-stranded RNA was inhibited by 1. Manoalide (1) also has the ability to inhibit the ATPase activity of human DHX36/RHAU, a putative RNA helicase. Taken together, we conclude that 1 inhibits the ATPase, RNA binding, and helicase activities of NS3 by targeting the helicase core domain conserved in both HCV NS3 and DHX36/RHAU.

Inhibition of Hepatitis C Virus Replication and Viral Helicase by Ethyl Acetate Extract of the Marine Feather Star Alloeocomatella polycladia

Hepatitis C virus (HCV) is a causative agent of acute and chronic hepatitis, leading to the development of hepatic cirrhosis and hepatocellular carcinoma. We prepared extracts from 61 marine organisms and screened them by an in vitro fluorescence assay targeting the viral helicase (NS3), which plays an important role in HCV replication, to identify effective candidates for anti-HCV agents. An ethyl acetate-soluble fraction of the feather star Alloeocomatella polycladia exhibited the strongest inhibition of NS3 helicase activity, with an IC50 of 11.7 µg/mL. The extract of A. polycladia inhibited interaction between NS3 and RNA but not ATPase of NS3. Furthermore, the replication of the replicons derived from three HCV strains of genotype 1b in cultured cells was suppressed by the extract with an EC50 value of 23 to 44 µg/mL, which is similar to the IC50 value of the NS3 helicase assay. The extract did not induce interferon or inhibit cell growth. These results suggest that the unknown compound(s) included in A. polycladia can inhibit HCV replication by suppressing the helicase activity of HCV NS3. This study may present a new approach toward the development of a novel therapy for chronic hepatitis C.

Estimation of single-nucleotide polymorphism allele frequency by alternately binding probe competitive polymerase chain reaction

Estimation of single-nucleotide polymorphism (SNP) allele frequency in pooled DNA samples is a promising approach to clarify the relationships between SNPs and diseases. Here, we present a simple, accurate, and cost-effective method for estimating SNP allele frequency, called alternately binding probe (ABProbe) competitive polymerase chain reaction (ABC-PCR) that entails no expensive devices for real-time fluorescence measurement and complex post-PCR steps. We prepared DNA pools of PCR products derived from homozygous samples of three different SNPs (ALDH2, GNB3, and HTR2A) in different portions, and the allele frequencies of these samples were estimated by ABC-PCR. Two alleles were coamplified by PCR with a fluorescent probe that binds to either alleles, and then fluorescence intensity was measured using a simple fluorometer. The ratio of the two alleles was calculated from the fluorescence intensity of the probe at the end-point. The estimated allele frequencies strongly correlated to the expected ratios for all three SNPs with high accuracy. When the allele frequencies were more than 5%, the relative standard deviations (R.S.D.s) of ABC-PCR were less than 20%. Moreover, we also confirmed that this method was applicable to SNP genotyping.

Cholesterol sulfate as a potential inhibitor of hepatitis C virus NS3 helicase.

Abstract Hepatitis C virus nonstructural protein 3 (NS3) helicase is a promising target for developing new therapeutics. In this study, we identified cholesterol sulfate (CS) as a novel NS3 helicase inhibitor (IC50 = 1.7 ± 0.2 µM with a Hill coefficient of 3.9) by screening the extracts from marine organisms. The lack of the sulfate group, sterol structure or alkyl side chain of CS diminished the inhibition, suggesting that an anion binding and hydrophobic region in NS3 may be a target site of CS. It was further found that CS partly inhibits NS3–RNA binding activity, but exerted no or less inhibition against ATPase and serine protease activities. Moreover, we demonstrated that CS probably does not bind to RNA. Our findings suggest that CS may inhibit NS3 helicase not by abolishing the other NS3 activities but by inducing conformational changes via interaction with possible allosteric sites of NS3.

Inhibition of Both Protease and Helicase Activities of Hepatitis C Virus NS3 by an Ethyl Acetate Extract of Marine Sponge Amphimedon sp

Combination therapy with ribavirin, interferon, and viral protease inhibitors could be expected to elicit a high level of sustained virologic response in patients infected with hepatitis C virus (HCV). However, several severe side effects of this combination therapy have been encountered in clinical trials. In order to develop more effective and safer anti-HCV compounds, we employed the replicon systems derived from several strains of HCV to screen 84 extracts from 54 organisms that were gathered from the sea surrounding Okinawa Prefecture, Japan. The ethyl acetate-soluble extract that was prepared from marine sponge Amphimedon sp. showed the highest inhibitory effect on viral replication, with EC50 values of 1.5 and 24.9 µg/ml in sub-genomic replicon cell lines derived from genotypes 1b and 2a, respectively. But the extract had no effect on interferon-inducing signaling or cytotoxicity. Treatment with the extract inhibited virus production by 30% relative to the control in the JFH1-Huh7 cell culture system. The in vitro enzymological assays revealed that treatment with the extract suppressed both helicase and protease activities of NS3 with IC50 values of 18.9 and 10.9 µg/ml, respectively. Treatment with the extract of Amphimedon sp. inhibited RNA-binding ability but not ATPase activity. These results suggest that the novel compound(s) included in Amphimedon sp. can target the protease and helicase activities of HCV NS3.

Identification of Hydroxyanthraquinones as Novel Inhibitors of Hepatitis C Virus NS3 Helicase.

Hepatitis C virus (HCV) is an important etiological agent of severe liver diseases, including cirrhosis and hepatocellular carcinoma. The HCV genome encodes nonstructural protein 3 (NS3) helicase, which is a potential anti-HCV drug target because its enzymatic activity is essential for viral replication. Some anthracyclines are known to be NS3 helicase inhibitors and have a hydroxyanthraquinone moiety in their structures; mitoxantrone, a hydroxyanthraquinone analogue, is also known to inhibit NS3 helicase. Therefore, we hypothesized that the hydroxyanthraquinone moiety alone could also inhibit NS3 helicase. Here, we performed a structure–activity relationship study on a series of hydroxyanthraquinones by using a fluorescence-based helicase assay. Hydroxyanthraquinones inhibited NS3 helicase with IC50 values in the micromolar range. The inhibitory activity varied depending on the number and position of the phenolic hydroxyl groups, and among different hydroxyanthraquinones examined, 1,4,5,8-tetrahydroxyanthraquinone strongly inhibited NS3 helicase with an IC50 value of 6 µM. Furthermore, hypericin and sennidin A, which both have two hydroxyanthraquinone-like moieties, were found to exert even stronger inhibition with IC50 values of 3 and 0.8 µM, respectively. These results indicate that the hydroxyanthraquinone moiety can inhibit NS3 helicase and suggest that several key chemical structures are important for the inhibition.