Biomedical subjects

Hideo Akashi

Publications and source records attributed to Hideo Akashi.

7 recordsLinked to original sources

PubMed2005

Escape from the interferon response associated with RNA interference using vectors that encode long modified hairpin-RNA.

In mammalian cells, siRNAs have been used to induce RNA interference (RNAi) in an attempt to prevent nonspecific effects (including the interferon (IFN) response) which are caused by long double-stranded RNAs (dsRNAs) of more than 30 bp. In this report, we describe a novel and simple strategy for avoiding activation of the IFN response by dsRNA. We show that modified hairpin-RNAs (mhRNAs) of more than 100 bp, with multiple specific point-mutations within the sense strand and transcribed from the U6 or tRNA(Val) promoters, can cause RNAi without inducing the IFN pathway genes. Moreover, we demonstrate that the 50-bp mhRNA vector could effectively suppress the replication of multiple hepatitis C viruses (the genomes of which differ slightly, thus the 21-bp siRNA vector failed to suppress one of them). Our findings should enhance the exploitation of RNAi in mammalian cells, especially in the field of RNAi therapy against pathogenic viruses.

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PubMed2005

Analysis of double-stranded RNA-induced apoptosis pathways using interferon-response noninducible small interfering RNA expression vector library.

We have developed an original vector library that allowed us to exploit the phenomenon of RNA interference but also allowed us to avoid the confounding effects of the interferon response. In the present work, we used our library of small interfering RNA expression vectors to examine the genes involved in apoptosis that was induced by double-stranded RNA. To our surprise, screening of our library revealed two novel double-stranded RNA-induced apoptotic pathways, a JNK/SAPK-mediated mitochondrial pathway and an ERK2-related pathway, both of which appeared to be independent of the serine-threonine protein kinase-dependent caspase pathway. We also found that MST2 and protein kinase Calpha both activated the pro-apoptotic signal mediated by ERK2. The results of our screening analysis suggested the utility of large scale screenings with libraries of small interfering RNA expression vectors.

Apoptosis↗

PubMed2005

Novel methods for expressing RNA interference in human cells.

RNA interference (RNAi) is a conserved process in which a double-stranded RNA (dsRNA) induces sequence-specific gene silencing. Recent developments in the use of the 21-nt small interfering RNA (siRNA) have allowed the specific degradation of mRNA without induction of nonspecific effects in mammalian cells. RNAi provides a method for knocking down genes of interest and a powerful tool for studies on gene functions in various organisms. Although many vector-based siRNA expression systems have been developed for production of siRNAs in mammalian cells, many technical issues for an effective production of siRNAs still need to be resolved. In this chapter, we describe methods for construction of genetically stable and highly active siRNA expression systems and also mention some strategies to overcome serious technical problems.

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PubMed2005

Gene discovery by ribozyme and siRNA libraries.

Catalytic RNAs, also known as ribozymes, can be engineered to optimize their activities in the intracellular environment. The introduction of a library of active ribozymes into cells, and the subsequent screening for phenotypic changes, allows the rapid identification of gene function. For the determination of gene function, ribozyme technology complements another RNA-based tool that is based on libraries of small interfering RNAs.

Animals↗

PubMed2005

Chemistry-based RNA technologies: demonstration of usefulness of libraries of ribozymes and short hairpin RNAs (shRNAs).

Mechanism of action of hammerhead ribozymes has been investigated and their intracellular activities have been improved. Based on the improved ribozymes and more recently discovered natural RNAi, we have created libraries of both ribozymes and short hairpin RNAs (shRNAs). The introduction of a library of active ribozymes or shRNAs into cells, and the subsequent screening for phenotypic changes, allows the rapid identification of gene function.

Animals↗

PubMed2004

RNAi expression vectors in plant cells.

Suppression by double-stranded RNA (dsRNA) of expression of a target gene is known as RNA interference (RNAi). Tobacco BY-2 cell suspension has been used as a model cultured plant cell, because it is possible to produce populations of tobacco BY-2 cell suspensions that are uniform and divide synchronously for functional gene analysis. Here, we describe a method to induce RNAi by introducing a hairpin-type dsRNA expression vector into BY-2 cells via electroporation. This methodology should facilitate the analysis of individual gene function in plant cells.

Animals↗

PubMed2002

Enhancement in the cleavage activity of a hammerhead ribozyme by cationic comb-type polymers and an RNA helicase in vitro.

The activity of a hammerhead ribozyme (Rz) in vivo depends on several factors, such as abundance, stability, and accessibility of Rz to its target mRNA. Among these factors, accessibility is believed to be the rate-limiting factor for Rz-mediated cleavage in vivo. As Rz and its substrate RNA are negatively charged, we examined whether cellular RNA-interacting proteins or artificial polycations might improve the accessibility of Rz to its substrate RNA. Specifically, we examined the effects of two kinds of cationic comb-type copolymer, alphaPLL-g-Dex, and a cellular RNA helicase on the accessibility of Rz to a model structured RNA in vitro. The cleavage activity of Rz was slightly enhanced by alphaPLL-g-Dex, probably due to an acceleration of the association/dissociation rate. And also, the RNA helicase-bound hybrid-Rz could cleave the target substrate at a significantly higher rate due to its unwinding activity for the duplex RNA substrate. These approaches should be useful in the development of efficient gene-inactivating reagents in the post-genomic era.

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