Application of RNA interference to study stem cell function: current status and future perspectives

RNA interference is a mechanism displayed by most eukaryotic cells to rid themselves of foreign double-stranded RNA molecules. In the six years since the initial report, RNA interference has now been demonstrated to function in mammalian cells to alter gene expression, and has been used as a means for genetic discovery as well as a possible strategy for genetic correction. An equally popular topic over the past six years has been the proposal to utilize embryonic stem cells or adult stem cells as cell-based therapies for human diseases. The aim of this review is to provide a general overview of how RNA interference suppresses gene expression and to examine some published RNA interference approaches that have resulted in changes in stem cell function and suggest the possible clinical relevance of this work.     

RNA干扰引起的非特异性免疫反应及其机制研究探讨

基因的表达失控是疾病发生的主要原因之一,干扰靶基因的表达可能成为有效的治疗手段。RNA干扰技术是近年兴起的基因调控干预方法,其基础,特别是应用研究极受关注,人们期待RNA干扰能成为肿瘤、病毒感染等难治疾病的临床治疗手段。然而,这一新兴技术在应用研究过程中显现出诸多问题,如细胞毒性、引起机体非特异性反应等等。就RNA干扰引起的非特异性免疫反应展开综述,探讨其机制,期望为RNA干扰的应用研究提供一些思考。     

丝状真菌中的RNA干扰及其应用技术

RNA干扰是真核生物中相对保守的一种基因特录后表达调控机制,它通过双链RNA介导细胞内mRNA发生特异性降解或翻译抑制,从而调控靶基因的表达.对丝状真菌中RNA压制和减数分裂沉默等现象的研究表明,与动、植物一样,丝状真菌中也存在RNA干扰现象.通过对RNA压制缺失突变株和减教分裂沉默缺失突变株的一系列分子生物学研究,获得了与之密切相关的一系列蛋白,而这些蛋白在结构和功能上与动、植物RNA干扰途径的蛋白高度相似,这些结果证明了丝状真菌中的RNA存在干扰现象.RNA干扰技术作为丝状真菌分子生物学研究或遗传改造的工具具有特殊的意义,因为丝状真菌具有多核和发生非同源重组频率高的特点,难以用基因敲除手段进行改造.系统地介绍丝状真菌中的RNA干扰途径以及使用RNA干扰对真菌进行遗传改造的方法.     

RNA interference in the pathogenic fungus Cryptococcus neoformans

Cryptococcus neoformans is a pathogenic fungus responsible for serious disease in immunocompromised individuals. This organism has recently been developed as an experimental system, with initiation of a genome project among other molecular advances. However, investigations of Cryptococcus are hampered by the technical difficulty of specific gene replacements. RNA interference, a process in which the presence of double-stranded RNA homologous to a gene of interest results in specific degradation of the corresponding message, may help solve this problem. We have shown that expression of double-stranded RNA corresponding to portions of the cryptococcal CAP59 and ADE2 genes results in reduced mRNA levels for those genes, with phenotypic consequences similar to that of gene disruption. The two genes could also be subjected to simultaneous interference through expression of chimeric double-stranded RNA. Specific modulation of protein expression through introduction of double-stranded RNA thus operates in C. neoformans, which is the first demonstration of this technique in a fungal organism. Use of RNA interference in Cryptococcus should allow manipulation of mRNA levels for functional analysis of genes of interest and enable efficient exploration of genes discovered by genome sequencing.     

Double-stranded RNA mediates selective gene silencing of protein phosphatase type 1 delta isoform in HEK-293 cells

The reversible phosphorylation of proteins mediates cellular signals in eukaryotic cells. RNA interference inhibits the expression of genes and proteins in a sequence-specific manner and provides a tool to study the functions of target molecules. The effect of RNA interference on protein phosphatase isoforms in HEK-293 cells was examined. Protein phosphatase 1 delta (PP1delta) sequence-specific double-stranded RNA (dsRNA) inhibited mRNA and protein expression of the PP1delta. This RNA interference did not affect the expression of lphaand gamma1 isoforms of PP1. Transfection of antisense RNA specific for PP1delta also suppressed the expression of PP1delta. It was further demonstrated by an in vitro RNA cleavage assay that extracts of HEK-293 cells catalyzed the processing of dsRNA. This cell line had much stronger mRNA expression of Dicer, an RNase III-like enzyme, than did human osteoblastic MG63 cells. The present results show that RNA interference is a useful tool to distinguish between PP1 isoforms.     

Killing the messenger: short RNAs that silence gene expression

Short interfering RNAs can be used to silence gene expression in a sequence-specific manner in a process that is known as RNA interference. The application of RNA interference in mammals has the potential to allow the systematic analysis of gene expression and holds the possibility of therapeutic gene silencing. Much of the promise of RNA interference will depend on the recent advances in short-RNA-based silencing technologies.     

RNA干扰与染色质沉默——生物体内精密的网络调控机制

基因表达受不同层次的调控.RNA干扰通过产生双链小RNA诱导同源mRNA序列降解,从而在转录后抑制特定基因的表达.最新的研究成果显示：RNA干扰产生的双链小RNA可通过与染色质中的重复序列DNA及组蛋白甲基化酶相互作用,引起组蛋白H3 Lys9的甲基化,进一步与异染色质形成相关蛋白结合,导致染色质沉默.综述了RNA干扰,小RNA,组蛋白修饰,染色质沉默及基因表达调控之间存在着精密的网络调控机制.     

RNA interference to target lipid disorders

PURPOSE OF REVIEW: This review focuses on proof-of-principle experiments providing validation of new targets for the development of RNA interference-based therapeutics for dyslipidemia. RECENT FINDINGS: Over the past few years, RNA interference has become an accepted approach to manipulate gene expression in mammalian systems. Advantage has been taken of the relative tissue specificity of adenovirus for liver, and the genetic specificity of short hairpin RNA-mediated RNA interference to create liver-specific downregulation of different genes. A different approach to target liver has been through the administration of chemically modified short interfering RNAs. For example, apolipoprotein B messenger RNA has been silenced in liver and jejunum resulting in decreased plasma levels of apolipoprotein B and total cholesterol. SUMMARY: RNA interference has aroused great interest as a powerful experimental tool and a potential therapeutic strategy. Successful animal studies indicate that RNA interference might be useful for the treatment of various human diseases. Clinical studies will soon begin to assess the use of this new class of therapeutics to treat dyslipidemia.     

Double-stranded RNA Mediates Selective Gene Silencing of Protein Phosphatase Type 1 Delta Isoform in HEK-293 Cells

The reversible phosphorylation of proteins mediates cellular signals in eukaryotic cells. RNA interference inhibits the expression of genes and proteins in a sequence-specific manner and provides a tool to study the functions of target molecules. The effect of RNA interference on protein phosphatase isoforms in HEK-293 cells was examined. Protein phosphatase 1 delta (PP1δ) sequence-specific double-stranded RNA (dsRNA) inhibited mRNA and protein expression of the PP18. This RNA interference did not affect the expression of α and γ1 isoforms of PP1. Transfection of antisense RNA specific for PP1δ also suppressed the expression of PP1δ. It was further demonstrated by an in vitro RNA cleavage assay that extracts of HEK-293 cells catalyzed the processing of dsRNA. This cell line had much stronger mRNA expression of Dicer, an RNase III-like enzyme, than did human osteoblastic MG63 cells. The present results show that RNA interference is a useful tool to distinguish between PP1 isoforms.     

Differential RNA interference: replacement of endogenous with recombinant low density lipoprotein receptor-related protein (LRP)

The interpretation of experiments involving the overexpression of a recombinant cDNA is often hampered by the interference of mRNA expression from the endogenous gene locus. Unless cell lines from naturally occurring mutations or knockout mice are available, difficult and time-consuming gene targeting techniques are required to inhibit endogenous gene expression. Using a method we refer to as "differential RNA interference" we demonstrate that RNA interference can be used to selectively suppress endogenous gene expression without affecting the expression of a co-transfected recombinant version of the same protein. Functional analyses of recombinant low density lipoprotein receptor-related protein (LRP) to study its involvement in lipid metabolism have been shown to be extremely difficult due to its large cDNA and the unavailability of suitable LRP-deficient cell lines. We constructed an expression vector containing the full-length coding sequence of human LRP fused to EGFP and a vector expressing small hairpin RNA directed against the 3'-untranslated region of the wild-type human LRP mRNA (LRP-shRNA). When overexpressed, EGFP-tagged LRP colocalizes with endogenous LRP and stimulates the uptake of LRP ligands. Overexpression of LRP-shRNA vectors significantly inhibits LRP expression, as judged by quantitative RT-PCR, Western blot and immunofluorescence analysis, and it dramatically decreases receptor-associated protein (RAP) uptake. Finally, co-transfection of EGFP-LRP and LRP-shRNA vectors demonstrates selective inhibition of endogenous LRP expression without affecting simultaneous expression of recombinant LRP protein. Thus, utilization of "differential RNA interference" provides a new experimental approach to selectively study the function of any recombinant protein in any given cell line without interference of endogenous protein expression.     

RNA干涉在纤毛虫中的研究进展

RNA干涉是dsRNA介导的基因沉默现象,本文简要介绍了其作用的机制和生物学意义,重点阐述了RNA干涉在原生动物纤毛虫中的发现与应用,比较了RNA干涉与纤毛虫大核基因组重排机理的异同,并对RNA干涉在纤毛虫中传输的技术途径-RNAi喂饲法的原理也做了详细的介绍。     

RNA干涉现象的发现与研究进展——2006年诺贝尔生理学或医学奖简介

RNA干涉现象以一种非常明确的方式抑制了基因表达,对于基因表达的调控、病毒感染的防护、控制跳跃基因具有重要的意义.它已被作为一种强大的“基因沉默”技术被用于全球的实验室,而且,会推动新的医疗技术的出现.     

Gene silencing with RNA interference in the human pathogenic fungus Aspergillus fumigatus

Aspergillus fumigatus is an opportunistic pathogenic fungus which causes fatal invasive aspergillosis among immunocompromised patients. To obtain a better understanding of the key elements involved in A. fumigatus virulence and to identify possible drug targets, it is necessary to be able to generate gene-deletion strains. Unfortunately, the molecular techniques available do not include a rapid method to disrupt and identify essential genes. RNA interference, a process in which the presence of double-stranded RNA homologous to a gene of interest results in specific degradation of the corresponding message, has been successfully tested on A. fumigatus. We have shown that expression of double stranded RNA corresponding to portions of the ALB1/PKSP and FKS1 genes results in reduced mRNA levels for those genes, with phenotypic consequences similar to that of gene disruption. The two genes could also be subjected to simultaneous interference through expression of chimeric double-stranded RNA. Use of RNA interference in Aspergillus will allow easier examination of the phenotypic consequences of reducing expression of a gene of interest, especially for essential genes.     

RNA interference and ovarian functions

RNA interference, a recently discovered new mechanism controlling gene expression via small RNAs, was shown to be involved in characterization and control of basic ovarian cell functions. The main classes of small RNAs, as well as their expression in ovaries have been described. Furthermore, the successful application of RNA interference for study and control of basic ovarian functions (proliferation, apoptosis, secretory activity, luteogenesis, oocyte maturation, and related ovarian cell malignant transformation) and production of recombinant proteins have been demonstrated. Application of RNA interference in reproductive biology and medicine can be successful in two main areas: (1) characterization and prediction of physiological and pathological state (association between particular small RNA and physiological or pathological processes), (2) application of small RNAs for regulation of reproductive processes and treatment of reproductive disorders or their particular indexes. Problems of improvement of small RNA delivery to target ovarian cells and potent RNA interference‐related approaches for treatment of ovarian disorders (especially of ovarian cancer) have been discussed. J. Cell. Physiol. 225: 354–363, 2010. © 2010 Wiley‐Liss, Inc.     

Insights into RNA virus mutant spectrum and lethal mutagenesis events: replicative interference and complementation by multiple point mutants

RNA virus behavior can be influenced by interactions among viral genomes and their expression products within the mutant spectra of replicating viral quasispecies. Here, we report the extent of interference of specific capsid and polymerase mutants of foot-and-mouth disease virus (FMDV) on replication of wild-type (wt) RNA. The capsid and polymerase mutants chosen for this analysis had been characterized biochemically and structurally. Upon co-electroporation of BHK-21 cells with wt RNA and a tenfold excess of mutant RNA, some mutants displayed strong interference (<10% of progeny production by wt RNA alone), while other mutants did not show detectable interference. The capacity to interfere required an excess of mutant RNA and was associated with intracellular replication, irrespective of the formation of infectious particles by the mutant virus. The extent of interference did not correlate with the known types and number of interactions involving the amino acid residue affected in each mutant. Synergistic interference was observed upon co-electroporation of wt RNA and mixtures of capsid and polymerase mutants. Interference was specific, in that the mutants did not affect expression of encephalomyocarditis virus RNA, and that a two nucleotide insertion mutant of FMDV expressing a truncated polymerase did not exert any detectable interference. The results support the lethal defection model for viral extinction by enhanced mutagenesis, and provide further evidence that the population behavior of highly variable viruses can be influenced strongly by the composition of the quasispecies mutant spectrum as a whole.     

Vector and parameters for targeted transgenic RNA interference in Drosophila melanogaster

The conditional expression of hairpin constructs in Drosophila melanogaster has emerged in recent years as a method of choice in functional genomic studies. To date, upstream activating site-driven RNA interference constructs have been inserted into the genome randomly using P-element-mediated transformation, which can result in false negatives due to variable expression. To avoid this problem, we have developed a transgenic RNA interference vector based on the phiC31 site-specific integration method.