Kimihiro Hino

CRISPRdirect: software for designing CRISPR/Cas guide RNA with reduced off-target sites

Summary: CRISPRdirect is a simple and functional web server for selecting rational CRISPR/Cas targets from an input sequence. The CRISPR/Cas system is a promising technique for genome engineering which allows target-specific cleavage of genomic DNA guided by Cas9 nuclease in complex with a guide RNA (gRNA), that complementarily binds to a ∼20 nt targeted sequence. The target sequence requirements are twofold. First, the 5′-NGG protospacer adjacent motif (PAM) sequence must be located adjacent to the target sequence. Second, the target sequence should be specific within the entire genome in order to avoid off-target editing. CRISPRdirect enables users to easily select rational target sequences with minimized off-target sites by performing exhaustive searches against genomic sequences. The server currently incorporates the genomic sequences of human, mouse, rat, marmoset, pig, chicken, frog, zebrafish, Ciona, fruit fly, silkworm, Caenorhabditis elegans, Arabidopsis, rice, Sorghum and budding yeast. Availability: Freely available at http://crispr.dbcls.jp/. Contact: y-naito@dbcls.rois.ac.jp Supplementary information: Supplementary data are available at Bioinformatics online.

Inducible expression of microRNA-194 is regulated by HNF-1α during intestinal epithelial cell differentiation

Maintenance of the intestinal epithelium is based on well-balanced molecular mechanisms that confer the stable and continuous supply of specialized epithelial cell lineages from multipotent progenitors. Lineage commitment decisions in the intestinal epithelium system involve multiple regulatory systems that interplay with each other to establish the cellular identities. Here, we demonstrate that the microRNA system could be involved in intestinal epithelial cell differentiation, and that microRNA-194 (miR-194) is highly induced during this process. To investigate this inducible expression mechanism, we identified the genomic structure of the miR-194-2, -192 gene, one of the inducible class of miR-194 parental genes. Furthermore, we identified its transcriptional regulatory region that contains a consensus-binding motif for hepatocyte nuclear factor-1alpha (HNF-1alpha), which is well known as a transcription factor to regulate gene expression in intestinal epithelial cells. By chromatin immunoprecipitation assay and luciferase reporter analysis, we revealed that pri-miR-194-2 expression is controlled by HNF-1alpha, and its consensus binding region is required for the transcription of pri-miR-194-2 in vivo in an intestinal epithelial cell line, Caco-2. Our observations indicate that microRNA genes could be targets of lineage-specific transcription factors and that microRNAs are regulated by a tissue-specific manner in the intestinal epithelium. Therefore, our work suggests that induced expression of these microRNAs have important roles in intestinal epithelium maturation.

Efficient oligonucleotide-mediated degradation of nuclear noncoding RNAs in mammalian cultured cells

Recent large-scale transcriptome analyses have revealed that large numbers of noncoding RNAs (ncRNAs) are transcribed from mammalian genomes. They include small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), and longer ncRNAs, many of which are localized to the nucleus, but which have remained functionally elusive. Since ncRNAs are only known to exist in mammalian species, established experimental systems, including the Xenopus oocyte system and yeast genetics, are not available for functional analysis. RNA interference (RNAi), commonly used for analysis of protein-coding genes, is effective in eliminating cytoplasmic mRNAs, but not nuclear RNAs. To circumvent this problem, we have refined the system for knockdown of nuclear ncRNAs with chemically modified chimeric antisense oligonucleotides (ASO) that were efficiently introduced into the nucleus by nucleofection. Under optimized conditions, our system appeared to degrade at least 20 different nuclear ncRNA species in multiple mammalian cell lines with high efficiency and specificity. We also confirmed that our method had greatly improved knockdown efficiency compared with that of the previously reported method in which ASOs are introduced with transfection reagents. Furthermore, we have confirmed the expected phenotypic alterations following knockdown of HBII295 snoRNA and U7 snRNA, which resulted in a loss of site-specific methylation of the artificial RNA and the appearance of abnormal polyadenylated histone mRNA species with a concomitant delay of the cell cycle S phase, respectively. In summary, we believe that our system is a powerful tool to explore the biological functions of the large number of nuclear ncRNAs with unknown function.

Stability of miRNA 5′terminal and seed regions is correlated with experimentally observed miRNA-mediated silencing efficacy

MicroRNAs (miRNAs) are key regulators of sequence-specific gene silencing. However, crucial factors that determine the efficacy of miRNA-mediated target gene silencing are poorly understood. Here we mathematized base-pairing stability and showed that miRNAs with an unstable 5′ terminal duplex and stable seed-target duplex exhibit strong silencing activity. The results are consistent with the previous findings that an RNA strand with unstable 5′ terminal in miRNA duplex easily loads onto the RNA-induced silencing complex (RISC), and miRNA recognizes target mRNAs with seed-complementary sequences to direct posttranscriptional repression. Our results suggested that both the unwinding and target recognition processes of miRNAs could be proficiently controlled by the thermodynamics of base-pairing in protein-free condition. Interestingly, such thermodynamic parameters might be evolutionarily well adapted to the body temperatures of various species.

A-to-I editing in the miRNA seed region regulates target mRNA selection and silencing efficiency

Hydrolytic deamination of adenosine to inosine (A-to-I) by adenosine deaminases acting on RNA (ADARs) is a post-transcriptional modification which results in a discrepancy between genomic DNA and the transcribed RNA sequence, thus contributing to the diversity of the transcriptome. Inosine preferentially base pairs with cytidine, meaning that A-to-I modifications in the mRNA sequences may be observed as A-to-G substitutions by the protein-coding machinery. Genome-wide studies have revealed that the majority of editing events occur in non-coding RNA sequences, but little is known about their functional meaning. MiRNAs are small non-coding RNAs that regulate the expression of target mRNAs with complementarities to their seed region. Here, we confirm that A-to-I editing in the miRNA seed duplex globally reassigns their target mRNAs in vivo, and reveal that miRNA containing inosine in the seed region exhibits a different degree of silencing efficiency compared to the corresponding miRNA with guanosine at the same position. The difference in base-pairing stability, deduced by melting temperature measurements, between seed-target duplexes containing either C:G or I:C pairs may account for the observed silencing efficiency. These findings unequivocally show that C:G and I:C pairs are biologically different in terms of gene expression regulation by miRNAs.

Downregulation of Nipah Virus N mRNA Occurs through Interaction between Its 3′ Untranslated Region and hnRNP D

ABSTRACT Nipah virus (NiV) is a nonsegmented, single-stranded, negative-sense RNA virus belonging to the genus Henipavirus, family Paramyxoviridae. NiV causes acute encephalitis and respiratory disease in humans, is associated with high mortality, and poses a threat in southern Asia. The genomes of henipaviruses are about 18,246 nucleotides (nt) long, which is longer than those of other paramyxoviruses (around 15,384 nt). This difference is caused by the noncoding RNA region, particularly the 3′ untranslated region (UTR), which occupies more than half of the noncoding RNA region. To determine the function(s) of the NiV noncoding RNA region, we investigated the effects of NiV 3′ UTRs on reporter gene expression. The NiV N 3′ UTR (nt 1 to 100) demonstrated strong repressor activity associated with hnRNP D protein binding to that region. Mutation of the hnRNP D binding site or knockdown of hnRNP D resulted in increased expression of the NiV N 3′ UTR reporter. Our findings suggest that NiV N expression is repressed by hnRNP D through the NiV N 3′ UTR and demonstrate the involvement of posttranscriptional regulation in the NiV life cycle. To the best of our knowledge, this provides the first report of the functions of the NiV noncoding RNA region.

Official Crime Rates and Residents’ Sense of Security Across Neighborhoods in Tokyo, Japan

The aim of this study was to investigate, in consideration of individual attributes and neighborhood-level social capital, the association between official crime rates and sense of neighborhood security among residents in the 23 wards of Tokyo, Japan, using data obtained from a national questionnaire survey and police statistics on crime for 511 neighborhoods. We found that crime rates affected residents’ sense of security differently according to the type of crime committed and the spatial scale. Regarding individual attributes, sense of security among men and those aged 35 to 49 years was in line with the actual property crime rate, whereas that among women and the elderly was in line with the actual violent crime rate. In addition, even when controlling for social capital, which had a strong positive effect on residents’ sense of security, and individual attributes, all crime rates except that for violent crime were significantly related to residents’ sense of security in their neighborhood.

Effects of Green Curtains to Improve the Living Environment

A green curtain means vertical planting of vines over a building surface. It is expected to improve indoor thermal environment of the building in summer, because it has a number of leaves shading a wall surface and windows of the building. However, it is also supposed to be obstacles to natural ventilation. Some experiments were done to confirm these environmental performances of green curtains. Model green curtains for the experiments were made of bitter gourd vines. A questionnaire survey for green curtain users was also carried out in Hamamatsu, Japan, to collect data about the amount of energy saved by green curtains and about the effect of green curtains on natural ventilation in residential houses. Results of the experiments and the survey indicate obvious effects of green curtains on indoor thermal environment.

Characteristics of fear of crime in evacuation shelters after the Great East Japan Earthquake

ABSTRACT After the Great East Japan Earthquake in March 2011, numerous evacuees had to stay in evacuation shelters such as school gymnasiums. In general, after a disaster, ensuring the safety and security of evacuees in evacuation shelters is a serious problem. Consequently, many of these evacuees feared that they would be victims of crime in the shelters or that their evacuated homes would be burgled. To examine what factors evoke a general fear of crime in shelters and burglary of evacuated homes, we conducted an online questionnaire on 300 evacuees in Miyagi Prefecture who had stayed at such shelters. From a factor analysis of fear of 16 specific kinds of crime in shelters, we extracted two factors representing fear of property and female-related crime. We then found that a general fear of crime in shelters was associated with being young and a fear of female-related crime, while a fear of burglary of evacuated homes was associated with being female, owning a detached house, and fear of property crime. Based on these results, we provide recommendations aiming to help reduce the fear of crime in shelters and burglary of evacuated homes.

Associations between seasonal meteorological conditions and the daily step count of adults in Yokohama, Japan: Results of year-round pedometer measurements in a large population

Peoples year-round interpersonal step count variations according to meteorological conditions are not fully understood, because complete year-round data from a sufficient sample of the general population are difficult to acquire. This study examined the associations between meteorological conditions and objectively measured step counts using year-round data collected from a large cohort (N = 24,625) in Yokohama, Japan from April 2015 to March 2016. Two-piece linear regression analysis was used to examine the associations between the monthly median daily step count and three meteorological indices (mean values of temperature, temperature-humidity index (THI), and net effective temperature (NET)). The number of steps per day peaked at temperatures between 19.4 and 20.7 °C. At lower temperatures, the increase in steps per day was between 46.4 and 52.5 steps per 1 °C increase. At temperatures higher than those at which step counts peaked, the decrease in steps per day was between 98.0 and 187.9 per 1 °C increase. Furthermore, these effects were more obvious in elderly than non-elderly persons in both sexes. A similar tendency was seen when using THI and NET instead of temperature. Among the three meteorological indices, the highest R2 value with step counts was observed with THI in all four groups. Both high and low meteorological indices discourage people from walking and higher values of the indices adversely affect step count more than lower values, particularly among the elderly. Among the three indices assessed, THI best explains the seasonal fluctuations in step counts.