RNA干涉在提高植物营养价值上的应用

RNA干涉(RNAi)是由具同源性的外源dsRNA引起的序列特异性转录后基因沉默现象,广泛存在于各种动、植物中。人类大多数疾病,像心脏病和癌症,都能够通过食用添加具有特殊营养成分养的饮食来预防。应用RNAi技术可为人和动物提高植物的营养价值。     

Strand-specific 5'-O-methylation of siRNA duplexes controls guide strand selection and targeting specificity

Small interfering RNAs (siRNAs) and microRNAs (miRNAs) guide catalytic sequence-specific cleavage of fully or nearly fully complementary target mRNAs or control translation and/or stability of many mRNAs that share 6-8 nucleotides (nt) of complementarity to the siRNA and miRNA 5' end. siRNA- and miRNA-containing ribonucleoprotein silencing complexes are assembled from double-stranded 21- to 23-nt RNase III processing intermediates that carry 5' phosphates and 2-nt overhangs with free 3' hydroxyl groups. Despite the structural symmetry of a duplex siRNA, the nucleotide sequence asymmetry can generate a bias for preferred loading of one of the two duplex-forming strands into the RNA-induced silencing complex (RISC). Here we show that the 5'-phosphorylation status of the siRNA strands also acts as an important determinant for strand selection. 5'-O-methylated siRNA duplexes refractory to 5' phosphorylation were examined for their biases in siRNA strand selection. Asymmetric, single methylation of siRNA duplexes reduced the occupancy of the silencing complex by the methylated strand with concomitant elimination of its off-targeting signature and enhanced off-targeting signature of the phosphorylated strand. Methylation of both siRNA strands reduced but did not completely abolish RNA silencing, without affecting strand selection relative to that of the unmodified siRNA. We conclude that asymmetric 5' modification of siRNA duplexes can be useful for controlling targeting specificity.     

TRBP alters human precursor microRNA processing in vitro

MicroRNAs play central roles in controlling gene expression in human cells. Sequencing data show that many miRNAs are produced at different levels and as multiple isoforms that can vary in length at their 5′ or 3′ ends, but the biogenesis and functional significance of these RNAs are largely unknown. We show here that the human trans-activation response (TAR) RNA binding protein (TRBP), a known molecular partner of the miRNA processing enzyme Dicer, changes the rates of pre-miRNA cleavage in an RNA-structure-specific manner. Furthermore, TRBP can trigger the generation of iso-miRNAs (isomiRs) that are longer than the canonical sequence by one nucleotide. We show that this change in miRNA processing site can alter guide strand selection, resulting in preferential silencing of a different mRNA target. These results implicate TRBP as a key regulator of miRNA processing and targeting in humans.     

dsRNA binding protein PACT/RAX in gene silencing,development and diseases

PACT （Protein kinase, interferon-inducible double stranded RNA dependent activator） and its murine ortholog RAX （PKR-associated protein X） were originally identified as a protein activator for the dsRNA-dependent, interferon-inducible protein kinase （PKR）. Endogenous PACT/RAX activates PKR in response to diverse stress signals such as serum starvation, and peroxide or arsenite treatment. PACT/RAX heterodimerized with PKR and activated it with its third motif in the absence of dsRNA. The activation of PKR leads to enhanced eIF2a phosphorylation followed by apoptosis or inhibition of growth. Besides the role of activating PKR, PACT is associated with a ~500 kDa complex that contains Dicer, hAgo2, and TRBP （TAR RNA binding protein） and it associates with Dicer to facilitate the production of small interfering RNA. PACT/RAX plays an important role in diverse physiological and pathological processes. Pact^-/- mice exhibit notable developmental abnormalities including microtia, with craniofacial ear, and hearing defects. Pact^-/- mice had smaller body sizes and fertility defects, both of which were caused by defective pituitary functions. It was found that dRAX disrupted fly embryos homozygous, displayed highly abnormal commissural axon structure of the central nervous system, and 70% of the flies homozygous for the mutant allele died prior to adulthood. Using high density SNP genotyping arrays, it was found that a mutation in PRKRA （the PACT/RAX gene） is the causative genetic mutation in DYT16, a novel autosomal recessive dystonia-parkinsonism syndrome in Brazilian patients.     

Short RNA duplexes guide sequence-dependent cleavage by human Dicer

Dicer is a member of the double-stranded (ds) RNA-specific ribonuclease III (RNase III) family that is required for RNA processing and degradation. Like most members of the RNase III family, Dicer possesses a dsRNA binding domain and cleaves long RNA duplexes in vitro. In this study, Dicer substrate selectivity was examined using bipartite substrates. These experiments revealed that an RNA helix possessing a 2-nucleotide (nt) 3'-overhang may bind and direct sequence-specific Dicer-mediated cleavage in trans at a fixed distance from the 3'-end overhang. Chemical modifications of the substrate indicate that the presence of the ribose 2'-hydroxyl group is not required for Dicer binding, but some located near the scissile bonds are needed for RNA cleavage. This suggests a flexible mechanism for substrate selectivity that recognizes the overall shape of an RNA helix. Examination of the structure of natural pre-microRNAs (pre-miRNAs) suggests that they may form bipartite substrates with complementary mRNA sequences, and thus induce seed-independent Dicer cleavage. Indeed, in vitro, natural pre-miRNA directed sequence-specific Dicer-mediated cleavage in trans by supporting the formation of a substrate mimic.     

siRNA selection criteria--statistical analyses of applicability and significance

RNA interference is a powerful tool for gene silencing, which is mediated by introducing siRNA. In the present study, statistical analyses of published siRNA selection criteria, the interpretation of some criteria and systematic searching for new criteria have been carried out for CGB siRNA and siRecords databases. The results of the analyses are as follows: (i) Our study supports the two-state model of the RNA-induced silencing complex (RISC). (ii) Stable 5'-S ends of a siRNA sequence, higher stability of the whole siRNA, and low breaking energy of siRNA duplex occurs in effective siRNA sequences. Also low internal stability of the 5'-AS terminus is preferred. (iii) Secondary structure can be successfully used as an RNAi selection criterion. (iv) Several published sequence criteria have been confirmed and also new criteria have been developed. (v) Also a Target Patterns criterion, which is comparable or better than the best known criteria, has been created.     

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。     

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。     

Suppressors of RNAi from plant viruses are subject to episodic positive selection

Viral suppressors of RNAi (VSRs) are proteins that actively inhibit the antiviral RNA interference (RNAi) immune response, providing an immune evasion route for viruses. It has been hypothesized that VSRs are engaged in a molecular ‘arms race’ with RNAi pathway genes. Two lines of evidence support this. First, VSRs from plant viruses display high sequence diversity, and are frequently gained and lost over evolutionary time scales. Second, Drosophila antiviral RNAi genes show high rates of adaptive evolution. Here, we investigate whether VSRs diversify faster than other genes and, if so, whether this is a result of positive selection, as might be expected in an arms race. By analysis of 12 plant RNA viruses, we show that the relative rate of protein evolution is higher for VSRs than for other genes, but that this is not attributable to pervasive positive selection. We argue that, because evolutionary time scales are extremely different for viruses and eukaryotes, it is improbable that viral adaptation (as measured by the ratio of non-synonymous to synonymous change) will be dominated by one-to-one coevolution with eukaryotes. Instead, for plant virus VSRs, we find strong evidence of episodic selection—diversifying selection that acts on a subset of lineages—which might be attributable to frequent shifts between different host genotypes or species.     

Systemic RNAi in western corn rootworm,Diabrotica virgifera virgifera,does not involve transitive pathways

Western corn rootworm (WCR, Diabrotica virgifera virgifera LeConte) is highly sensitive to orally delivered double‐stranded RNA (dsRNA). RNAi in WCR is systemic and spreads throughout the insect body. This raises the question whether transitive RNAi is a mechanism that functions in WCR to amplify the RNAi response via production of secondary siRNA. Secondary siRNA production is achieved through RNA‐dependent RNA polymerase (RdRP) activity in other eukaryotic organisms, but RdRP has not been identified in WCR and any other insects. This study visualized the spread of the RNAi‐mediated knockdown of Dv v‐ATPase C mRNA throughout the WCR gut and other tissues using high‐sensitivity branched DNA in situ hybridization. Furthermore, we did not detect either secondary siRNA production or transitive RNAi in WCR through siRNA sequence profile analysis. Nucleotide mismatched sequences introduced into either the sense or antisense strand of v‐ATPase C dsRNAs were maintained in siRNAs derived from WCR fed with the mismatched dsRNAs in a strand specific manner. The distribution of all siRNAs was restricted to within the original target sequence regions, which may indicate the lack of new dsRNA synthesis leading to production of secondary siRNA. Thus, the systemic spread of RNAi in WCR may be derived from the original dsRNA molecules taken up from the gut lumen. These results indicate that the initial dsRNA dose is important for a lethal systemic RNAi response in WCR and have implications in developing effective dsRNA traits to control WCR and in resistance management to prolong the durability of RNAi trait technology.     

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.     

Two molecular features contribute to the Argonaute specificity for the microRNA and RNAi pathways in C. elegans

In Caenorhabditis elegans, specific Argonaute proteins are dedicated to the RNAi and microRNA pathways. To uncover how the precise Argonaute selection occurs, we designed dsRNA triggers containing both miRNA and siRNA sequences. While dsRNA carrying nucleotides mismatches can only enter the miRNA pathway, a fully complementary dsRNA successfully rescues let-7 miRNA function and initiates silencing by RNAi. We demonstrated that RDE-1 is essential for RNAi induced by the perfectly paired trigger, yet is not required for silencing by the let-7 miRNA. In contrast, ALG-1/ALG-2 are required for the miRNA function, but not for the siRNA-directed gene silencing. Finally, a dsRNA containing a bulged miRNA and a perfectly paired siRNA can enter both pathways suggesting that the sorting of small RNAs occurs after that the dsRNA trigger has been processed by Dicer. Thus, our data suggest that the selection of Argonaute proteins is affected by two molecular features: (1) the structure of the small RNA duplex; and (2) the Argonautes specific characteristics.     

Stable knockdown of MYCN by lentivirus-based RNAi inhibits human neuroblastoma cells growth in vitro and in vivo

Neuroblastoma is the most common childhood solid tumor, yet current treatment approaches have not been able to effectively control this cancer. Amplification and overexpression of MYCN have been shown to be closely related with high risk and poor prognosis in neuroblastoma. This suggests that MYCN is an important target for the antitumor therapy. Recently, vector-based RNA interference (RNAi) systems have been successfully used to eliminate gene expression, but knockdown of MYCN by vector-based RNAi as a therapeutic model for neuroblastoma has not been fully established.In this study, we used a lentivirus vector-based RNAi approach which expresses short hairpin RNA (shRNA) to knockdown MYCN in neuroblastoma cell lines IMR-32 and LAN-1. Western blotting analysis showed that expressions of MYCN were efficiently downregulated after infection with MYCN shRNA expression vector. The stable suppression of MYCN expression induced differentiation and apoptosis in neuroblastoma cell lines. Furthermore, we demonstrated that these changes were associated with caspase-3 activation, p27 upregulation as well as Bcl-2 and MDM2 downregulation. Finally, we demonstrated that downregulation of MYCN expression significantly reduced colony formation in vitro and tumor growth in nude mice.Our data indicate that lentivirus vector-mediated silencing of MYCN in neuroblastoma cells could efficiently and significantly inhibit tumor growth both in vitro and in vivo. Therefore we demonstrate the therapeutic potential of lentivirus-delivered shRNA as a novel approach for treatment of neuroblastoma and other malignant tumors with MYCN overexpression.     

新型RNAi载体抑制HPV16E6基因在人宫颈癌细胞Caski中的表达

目的：利用自行构建的一种基于Semliki森林病毒的新型RNAi载体pSFV-RNAi Ready,验证将其用于短期高效沉默HPV16E6基因的效果。方法：以HPV16E6为靶标基因,设计并构建基于pSFV-RNAi Ready的重组质粒,分直接电击转染和病毒颗粒共培养两种方式转入人宫颈癌细胞株Caski,RT-PCR、Western blot检测HPV16E6表达水平。结果：重组质粒对HPV16E6沉默效果优于常规RNAi质料载体,接近化学合成小RNA,抑制率可高达90%以上,10天后效果仍然存在;结论：新型RNAi载体pSFV-RNAi Ready可较好地应用于特异高丰度靶基因的表达抑制,有望用于未来的科学研究或治疗应用。