昆虫RNAi通路及其核心元件的研究综述

RNAi作为分子生物学的一种重要技术,在昆虫基因功能和功能基因组研究中得到广泛应用,同时,有关昆虫RNAi的机制也受到了大家的关注。近年来的研究结果表明,昆虫RNAi的通路与其他动物相同,根据引起基因沉默的RNA分子的类型,可以分为siRNA、miRNA和piRNA 3种不同的通路。昆虫RNAi通路中的核心元件包括了:(1)行使切割作用的RNaseⅢ家族成员Drosha和Dicer;(2)用来降解目的 mRNA的Argonaute蛋白;(3)dsRNA结合蛋白Pasha、R2D2和Loquacious。了解昆虫RNAi的通路及其核心元件,有助于我们更好地理解昆虫RNAi的分子机制和改进实现RNAi的方法,对促进昆虫RNAi技术的研究及其在害虫防控中的应用具有指导意义。     

RNAi技术及在植物抗病毒研究中的应用

RNA干扰(RNA interference,RNAi)技术是一项基因沉默新技术,在抗病毒研究中,人为地将与病毒或宿主基因(宿主基因编码的蛋白质对病毒很重要而对宿主本身作用很小或不起作用)同源的双链RNA(double strand RNA,dsRNA)导入生物体内,引起与其同源的基因发生沉默,从而抑制病毒复制,达到抗病毒的目的。因此RNAi技术在抗病毒研究中倍受关注,并取得了显著成绩。主要对RNAi技术的相关知识以及在植物抗病毒中的应用进展作一综述。     

Transgenic RNAi: Accelerating and Expanding Reverse Genetics in Mammals

Reverse genetics in mammals has relied on gene targeting strategies and has mostly been limited to the mouse. Gene targeting through homologous recombination in mouse ES cells has drawbacks which include time, expense and complexity. Recently, a new approach has been developed based on RNA-interference (RNAi) which is simpler, faster, less expensive, and should be applicable to mammalian species other than mouse. The advent of RNAi is poised to accelerate the pace at which reverse genetics can be applied to study gene function in mammals.     

Minimizing variables among hairpin-based RNAi vectors reveals the potency of shRNAs

RNA interference (RNAi) is a cellular process regulating gene expression and participating in innate defense in many organisms. RNAi has also been utilized as a tool to query gene function and is being developed as a therapeutic strategy for several diseases. Synthetic small interfering (siRNAs) or expressed stem–loop RNAs (short-hairpin RNAs [shRNAs] or artificial microRNAs [miRNAs]) have been delivered to cultured cells and organisms to inhibit expression of a variety of genes. A persistent question in the field, however, is which RNAi expression system is most suitable for distinct applications. To date, shRNA- and artificial miRNA-based strategies have been compared with conflicting results. In prior comparisons, sequences required for efficient RNAi processing and loading of the intended antisense strand into the RNAi-induced silencing complex (RISC) were not considered. We therefore revisited the shRNA–miRNA comparison question. Initially, we developed an improved artificial miRNA vector and confirmed the optimal shRNA configuration by altering structural features of these RNAi substrates. Subsequently, we engineered and compared shRNA- and miRNA-based RNAi expression vectors that would be processed to yield similar siRNAs that exhibit comparable strand biasing. Our results demonstrate that when comparison variables are minimized, the shRNAs tested were more potent than the artificial miRNAs in mediating gene silencing independent of target sequence and experimental setting (in vitro and in vivo). In addition, we show that shRNAs are expressed at considerably higher levels relative to artificial miRNAs, thus providing mechanistic insight to explain their increased potency.     

Kinetic barriers and the role of topology in protein and RNA folding

This review compares the folding behavior of proteins and RNAs. Topics covered include the role of topology in the determination of folding rates, major folding events including collapse, properties of denatured states, pathway heterogeneity, and the influence of the mode of initiation on the folding pathway.     

Distinct nuclear and cytoplasmic machineries cooperatively promote the inheritance of RNAi in Caenorhabditis elegans

Epigenetic information can be inherited over multiple generations, which is termed as transgenerational epigenetic inheritance (TEI). Although the mechanism(s) of TEI remains poorly understood, noncoding RNAs have been demonstrated to play important roles in TEI. In many eukaryotes, double‐stranded RNA (dsRNA) triggers the silencing of cellular nucleic acids that exhibit sequence homology to the dsRNA via a process termed RNA interference (RNAi). In Caenorhabditis elegans, dsRNA‐directed gene silencing is heritable and can persist for a number of generations after its initial induction. During the process, small RNAs and the RNAi machinery mediate the initiation, transmission and re‐establishment of the gene silencing state. In this review, we summarise our current understanding of the underlying mechanism(s) of transgenerational inheritance of RNAi in C. elegans and propose that multiple RNAi machineries may act cooperatively to promote TEI.     

Long-term effects and parental RNAi in the blood feeder Rhodnius prolixus (Hemiptera; Reduviidae)

RNA interference (RNAi) has been widely employed as a useful alternative to study gene function in insects, including triatomine bugs. However, several aspects related to the RNAi mechanism and functioning are still unclear. The aim of this study is to investigate the persistence and the occurrence of systemic and parental RNAi in the triatomine bug Rhodnius prolixus. For such, the nitrophorins 1 to 4 (NP1-4), which are salivary hemeproteins, and the rhodniin, an intestinal protein, were used as targets for RNAi. The dsRNA for both molecules were injected separately into 3rd and 5th instar nymphs of R. prolixus and the knockdown (mRNA levels and phenotype) were progressively evaluated along several stages of the insect's life. We observed that the NP1-4 knockdown persisted for more than 7 months after the dsRNA injection, and at least 5 months in rhodniin knockdown, passing through various nymphal stages until the adult stage, without continuous input of dsRNA. The parental RNAi was successful from the dsRNA injection in 5th instar nymphs for both knockdown targets, when the RNAi effects (mRNA levels and phenotype) were observed at least in the 2nd instar nymphs of the F1 generation. However, the parental RNAi did not occur when the dsRNA was injected in the 3rd instars. The confirmation of the long persistence and parental transmission of RNAi in R. prolixus can improve and facilitate the utilization of this tool in insect functional genomic studies.     

慢病毒载体介导RNAi的研究进展

RNAi通过双链RNA的介导,特异性阻抑相关序列的表达,从而导致转录后水平的基因沉默.广泛存在于真菌、植物和动物等真核生物中.慢病毒载体是理想的真核细胞基因转移工具,被广泛应用于相关的RNAi研究领域,例如抗病毒研究、癌症及其治疗、遗传性疾病的治疗、基因治疗.现已发现,慢病毒载体能够介导组织特异、时间特异的RNAi,在疾病的基因靶向性治疗上必有广阔的前景.     

Small RNAs, RNAi and the Inheritance of Gene Silencing in Caenorhabditis elegans

Invasive nucleic acids such as transposons and viruses usually exhibit aberrant characteristics, e.g., unpaired DNA or abnormal double-stranded RNA. Organisms employ a variety of strategies to defend themselves by distinguishing self and nonself substances and disabling these invasive nucleic acids. Furthermore, they have developed ways to remember this exposure to invaders and transmit the experience to their descendants. The mechanism underlying this inheritance has remained elusive. Recent research has shed light on the initiation and maintenance of RNA-mediated inherited gene silencing. Small regulatory RNAs play a variety of crucial roles in organisms, including gene regulation, developmental timing, antiviral defense, and genome integrity, via a process termed as RNA interference (RNAi). Recent research has revealed that small RNAs and the RNAi machinery are engaged in establishing and promoting transgenerational gene silencing. Small RNAs direct the RNAi and chromatin modification machinery to the cognate nucleic acids to regulate gene expression and epigenetic alterations. Notably, these acquired small RNAs and epigenetic changes persist and are transmitted from parents to offspring for multiple generations. Thus, RNAi is a vital determinant of the inheritance of gene silencing and acts as a driving force of evolution.     

An Altered Method of Feeding RNAi That Knocks Down Multiple Genes Simultaneously in the Nematode Caenorhabditis elegans

In reverse genetics, RNA interference (RNAi) which is substitutable for gene-disruption, is an outstanding method for knockdown of a gene’s function. In Caenorhabditis elegans, feeding RNAi is most convenient, but this RNAi is not suitable for knockdown of multiple genes. Hence, we attempted to establish an efficient method of feeding RNAi for multiple knockdown. We produced bacteria yielding three distinct double-stranded RNAs bound to one another, and fed those bacteria to C. elegans. Quantitative RT-PCR and observation of phenotypes indicated that our method is much more efficient than the traditional one. Our method is useful for investigating genes’ functions in C. elegans.     

How RNAi machinery enters the world of telomerase

Human telomerase holoenzyme consists of the catalytic component TERT and the template RNA TERC. However, a network of accessory proteins plays key roles in its assembly, localization and stability. Defects in genes involved in telomerase biology affect the renewal of critical stem cell populations and cause disorders such as telomeropathies. Moreover, activation of telomerase in somatic cells allows neoplastic cells to proliferate indefinitely, thus contributing to tumorigenesis. For these reasons, identification of new players involved in telomerase regulation is crucial for the determination of novel therapeutic targets and biomarkers. In the very last years, increasing evidence describes components of the RNAi machinery as a new layer of complexity in human telomerase activity. In this review, we will discuss how AGO2 and other proteins which collaborate with AGO2 in RNAi pathway play a pivotal role in TERC stability and function.     

RNAi Transfection Results in Lipidome Changes

RNAi experiments are ubiquitously used in cell biology and are achieved by transfection of small interfering RNAs (siRNAs) into cells using a transfection reagent. These results in knock‐down of proteins of interest, and the phenotypic consequences are then analyzed. It is reported here that two common RNA interference (RNAi) transfection reagents, DharmaFECT 1 and INTERFERin, in mock transfections using non‐targeting siRNAs, cause alterations in the lipidome of HeLa cells. Some lipids change in response to both, presumably chemically different, transfection reagents, while other lipid species change only in response to one of the reagents. While the functional implications of these lipidomic alterations remain to be investigated, the authors' experiments suggest that it is important to use appropriate mock transfection controls during RNAi experiments, ideally complemented by an orthogonal perturbation, especially when investigating membrane‐associated phenomena.     

通过RNA干涉沉默SARS病毒的研究前景

RNA干涉(RNAi)是双链RNA介导的转录后基因沉默作用的重要机制之一。RNAi在后基因组时代的基因功能研究和药物开发中具有广阔应用前景。本综述了RNAi在抗SARS病毒药物研究中的应用前景。     

On the structural repertoire of pools of short, random RNA sequences

A detailed knowledge of the mapping between sequence and structure spaces in populations of RNA molecules is essential to better understand their present-day functional properties, to envisage a plausible early evolution of RNA in a prebiotic chemical environment and to improve the design of in vitro evolution experiments, among others. Analysis of natural RNAs, as well as in vitro and computational studies, show that certain RNA structural motifs are much more abundant than others, pointing out a complex relation between sequence and structure. Within this framework, we have investigated computationally the structural properties of a large pool (10⁸ molecules) of single-stranded, 35 nt-long, random RNA sequences. The secondary structures obtained are ranked and classified into structure families. The number of structures in main families is analytically calculated and compared with the numerical results. This permits a quantification of the fraction of structure space covered by a large pool of sequences. We further show that the number of structural motifs and their frequency is highly unbalanced with respect to the nucleotide composition: simple structures such as stem-loops and hairpins arise from sequences depleted in G, while more complex structures require an enrichment of G. In general, we observe a strong correlation between subfamilies—characterized by a fixed number of paired nucleotides—and nucleotide composition. Our results are compared to the structural repertoire obtained in a second pool where isolated base pairs are prohibited.     

Uncovering RNAi mechanisms in plants: biochemistry enters the foray

In plants, the RNA interference (RNAi) machinery responds to a variety of triggers including viral infection, transgenes, repeated elements and transposons. All of these triggers lead to silencing outcomes ranging from mRNA degradation to translational repression to chromatin remodeling. Thus, plants offer us a potentially unique opportunity to understand the full range of RNAi effector mechanisms. In this review, we discuss the recent developments in our understanding of plant RNAi mechanisms from a biochemical perspective.     

多靶向RNA干扰技术在基因治疗中的应用与前景

RNA干扰(RNA interference,RNAi)通过转录后基因沉默效应特异性抑制靶基因的表达,其沉默机制的高效性、特异性及稳定性使这项技术成为生物医学领域研究基因治疗的重要工具。阐述RNAi技术的特点和RNAi疗法的现状,特别是多靶小干扰RNA(small interference RNA,siRNA)目前的发展态势及其各种结构性修饰,通过使用这些结构修饰的siRNA提高基因沉默的效率,将有助于提高疗效。但该技术在广泛应用于临床之前,仍存在一些亟待解决的问题与面临的挑战,需进一步研究。