In vitro analysis of RNA interference in Drosophila melanogaster

Double-stranded RNA (dsRNA) triggers the destruction of mRNA sharing sequence with the dsRNA, a phenomenon termed RNA interference (RNAi). The dsRNA is converted by endonucleolytic cleavage into 21- to 23-nt small interfering RNAs (siRNAs), which direct a multiprotein complex, the RNA-induced silencing complex to cleave RNA complementary to the siRNA. RNAi can be recapitulated in vitro in lysates of syncytial blastoderm Drosophila embryos. These lysates reproduce all of the known steps in the RNAi pathway in flies and mammals. Here we explain how to prepare and use Drosophila embryo lysates to dissect the mechanism of RNAi.     

In vivo analysis of the RNA interference mechanism in Trypanosoma brucei

Flagellate protozoa of the family Trypanosomatidae, which includes various members of the genera Leishmania and Trypanosoma, are model systems for unicellular pathogens to study fundamentally important biological phenomena. Recently, ablation of gene expression by RNA interference (RNAi) has become the method of choice to study gene function in Trypanosoma brucei, an early divergent eukaryote that infects humans and animals. As has been shown in multicellular organisms, the RNAi mechanism in T. brucei involves processing of double-stranded RNA 24- to 26-nt RNAs, termed small interfering RNAs (siRNAs), which guide degradation of the target mRNA. In this article, we describe some of the methods we employ for the analysis of the RNAi mechanism in T. brucei with particular emphasis on detection, cloning, and fractionation of siRNAs and siRNA complexes.     

Preferential spreading of RNA silencing into the 3’ downstream region of the transgene in tobacco

The amplification mechanism of short interfering RNAs (siRNAs) along the transgene sequence exists in RNA interference (RNAi). The RNA-dependent RNA polymerase synthesizes complementary RNAs by using the transgene mRNA as a template, and the secondary siRNAs are generated from the outside of primary RNAi target. Four independent RNAi vectors which produced primary siRNAs against distinct regions of the tobacco endoplasmic reticulum ω-3 fatty acid desaturase gene (NtFAD3) were transiently expressed in leaves of theNtF4D3-overexpressed transgenic plants. Regardless of the RNAi vector used, the secondaryNtFAD3 siRNAs were generated preferentially from the 3’ downstream region of the transgene. Secondary siRNAs from the 5’ upstream region adjacent to the annealing site of primary siRNAs accumulated under the detection level. Our results suggest that different regulatory mechanisms are involved in the spreading of RNA silencing into 5’ upstream and 3’ downstream regions of the target sequence, respectively.     

Identification of sequence features that predict competition potency of siRNAs

Small interfering RNAs (siRNAs) specifically knock-down target mRNAs via RNA interference (RNAi) mechanism. During this process, introduction of excess amount of exogenous siRNAs could lead to the saturation of cellular RNAi machinery. One consequence of RNAi machinery saturation is the competition between two simultaneously introduced siRNAs, during which one siRNA loses gene silencing activity. Although competition phenomena have been well characterized, the molecular and sequence features of siRNAs that specify the competition potency remain poorly understood. Here, for the first time, we performed a large-scale siRNA competition potency analysis by measuring the competition potency of 56 different siRNAs and ranking them based on their competition potency. We have also established an algorithm to predict the competition potency of siRNAs based upon the conserved sequence features of strong and weak competitor siRNAs. The present study supports our hypothesis that the competition potency of siRNAs is specified by the 5′-half antisense sequence and provides a useful guideline to design siRNAs with minimal RNAi machinery saturation.     

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.     

Inhibition of hepatitis B virus replication in cultured cells and in vivo using 2′-O-guanidinopropyl modified siRNAs

Silencing hepatitis B virus (HBV) gene expression with exogenous activators of the RNA interference (RNAi) pathway has shown promise as a new mode of treating infection with the virus. However, optimizing efficacy, specificity, pharmacokinetics and stability of RNAi activators remains a priority before clinical application of this promising therapeutic approach is realised. Chemical modification of synthetic short interfering RNAs (siRNAs) provides the means to address these goals. This study aimed to assess the benefits of incorporating nucleotides with 2′-O-guanidinopropyl (GP) modifications into siRNAs that target HBV. Single GP residues were incorporated at nucleotide positions from 2 to 21 of the antisense strand of a previously characterised effective antiHBV siRNA. When tested in cultured cells, siRNAs with GP moieties at selected positions improved silencing efficacy. Stability of chemically modified siRNAs in 80% serum was moderately improved and better silencing effects were observed without evidence for toxicity or induction of an interferon response. Moreover, partially complementary target sequences were less susceptible to silencing by siRNAs with GP residues located in the seed region. Hydrodynamic co-injection of siRNAs with a replication-competent HBV plasmid resulted in highly effective knock down of markers of viral replication in mice. Evidence for improved efficacy, reduced off target effects and good silencing in vivo indicate that GP-modifications of siRNAs may be used to enhance their therapeutic utility.     

Increased siRNA duplex stability correlates with reduced off-target and elevated on-target effects

Argonaute (Ago) proteins form the core of RNA-induced silencing complexes (RISCs) and mediate small RNA-guided gene silencing. In RNAi, short interfering RNAs (siRNAs) guide RISCs to complementary target RNAs, leading to cleavage by the endonuclease Ago2. Noncatalytic Ago proteins, however, contribute to RNAi as well but cannot cleave target RNA and often generate off-target effects. Here we show that synthetic siRNA duplexes interact with all Ago proteins, but a functional RISC rapidly assembles only around Ago2. By stabilizing the siRNA duplex, we show that the noncatalytic Ago proteins Ago1, -3, and -4 can be selectively blocked and do not form functional RISCs. In addition, stabilized siRNAs form an Ago2-RISC more efficiently, leading to increased silencing activity. Our data suggest novel parameters for the design of siRNAs with selective activation of the endonuclease Ago2.     

Role of the Trypanosoma brucei HEN1 Family Methyltransferase in Small Interfering RNA Modification

Parasitic protozoa of the flagellate order Kinetoplastida represent one of the deepest branches of the eukaryotic tree. Among this group of organisms, the mechanism of RNA interference (RNAi) has been investigated in Trypanosoma brucei and to a lesser degree in Leishmania (Viannia) spp. The pathway is triggered by long double-stranded RNA (dsRNA) and in T. brucei requires a set of five core genes, including a single Argonaute (AGO) protein, T. brucei AGO1 (TbAGO1). The five genes are conserved in Leishmania (Viannia) spp. but are absent in other major kinetoplastid species, such as Trypanosoma cruzi and Leishmania major. In T. brucei small interfering RNAs (siRNAs) are methylated at the 3′ end, whereas Leishmania (Viannia) sp. siRNAs are not. Here we report that T. brucei HEN1, an ortholog of the metazoan HEN1 2′-O-methyltransferases, is required for methylation of siRNAs. Loss of TbHEN1 causes a reduction in the length of siRNAs. The shorter siRNAs in hen1^−/− parasites are single stranded and associated with TbAGO1, and a subset carry a nontemplated uridine at the 3′ end. These findings support a model wherein TbHEN1 methylates siRNA 3′ ends after they are loaded into TbAGO1 and this methylation protects siRNAs from uridylation and 3′ trimming. Moreover, expression of TbHEN1 in Leishmania (Viannia) panamensis did not result in siRNA 3′ end methylation, further emphasizing mechanistic differences in the trypanosome and Leishmania RNAi mechanisms.     

Competition potency of siRNA is specified by the 5'-half sequence of the guide strand

Small-interfering RNAs (siRNAs) execute specific cellular gene silencing by exploiting the endogenous RNA interference (RNAi) pathway. Therefore, excess amounts of siRNAs can saturate cellular RNAi machineries. Indeed, some siRNAs saturate the RNA-induced silencing complex (RISC) and competitively inhibit silencing by other siRNAs. However, the molecular feature of siRNAs that specifies competition potency has been undetermined. While previous reports suggested a correlation between the competition potency and silencing efficiency of siRNAs, we found that the silencing efficiency was insufficient to explain the competition potency. Instead, we show that the nucleotide sequence of the 5′-half of the guide strand determines the competition potency of an siRNA. Our finding provides important information for understanding the mechanistic basis of competition in combinatorial RNAi treatment.     

Synthesis and properties of double-stranded RNA-bindable oligodiaminogalactose derivatives conjugated with vitamin E

RNA interference (RNAi) is a gene-regulating system that is controlled by external short interfering RNAs (siRNAs). Sequence selective gene silencing by siRNA shows promise in clinical research. However, there have been few efficient methods for delivering siRNAs to target cells. In this study, we propose a novel type of RNA duplex-bindable molecule with an oligodiaminosaccharide structure. These 2,6-diamino-2,6-dideoxy-(1-4)-β-d-galactopyranose oligomers (oligodiaminogalactoses; ODAGals) conjugated with α-tocopherol (vitamin E; VE) or a VE analog were designed as novel siRNA-bindable molecules that can be utilized to deliver RNAi drugs to the liver. Among these compounds, the VE analog-bound ODAGal was suggested to bind to RNA duplexes without inhibiting RNAi activity.     

RNA干扰在疾病治疗方面的应用研究

RNA干扰是由双链RNA引起的序列特异的基因沉默现象。由于RNA干扰能在细胞组织及动物模型中沉默疾病相关基因,因此,RNA干扰也是各种疾病治疗的有效手段。在哺乳动物细胞内诱导RNA干扰可以通过导入小干扰RNA（siRNA）,或是以质粒、病毒为载体表达短的发夹RNA（shRNA）而实现。本文介绍了RNA干扰在疾病治疗方面的应用,并就其面临的挑战进行讨论。     

RNA interference induced by siRNAs modified with 4'-thioribonucleosides in cultured mammalian cells

Short interfering RNAs (siRNAs) variously modified with 4'-thioribonucleosides against the Photinus luciferase gene were tested for their induction of the RNA interference (RNAi) activity in cultured NIH/3T3 cells. Results indicated that modifications at the sense-strand were well tolerated for RNAi activity except for full modification with 4'-thioribonucleosides. However, the activity of siRNAs modified at the antisense-strand was dependent on the position and the number of modifications with 4'-thioribonucleosides. Since modifications of siRNAs with 4'-thioribonucleosides were well tolerated in RNAi activity compared with that of 2'-O-methyl nucleosides, 4'-thioribonucleosides might be potentially useful in the development of novel and effective chemically modified siRNAs.     

RNA沉默在植物生物逆境反应中的作用

RNA沉默是真核生物共有的基因表达调节机制和防御机制。在植物RNA沉默中, 一些小RNAs, 如微小 RNAs和小干扰RNAs, 在植物防御病毒、细菌或食草动物的反应中具有重要作用。为了抑制宿主的RNA沉默系统, 植物病毒或细菌进化出了在RNA沉默不同阶段起作用的病毒沉默抑制子或细菌沉默抑制子, 来克服寄主的RNA沉默反应。文章就植物RNA沉默、病毒沉默抑制子、细菌沉默抑制子及其相关防御反应的一些新进展做一概述。