A distinct small RNA pathway silences selfish genetic elements in the germline

In the Drosophila germline, repeat-associated small interfering RNAs (rasiRNAs) ensure genomic stability by silencing endogenous selfish genetic elements such as retrotransposons and repetitive sequences. Whereas small interfering RNAs (siRNAs) derive from both the sense and antisense strands of their double-stranded RNA precursors, rasiRNAs arise mainly from the antisense strand. rasiRNA production appears not to require Dicer-1, which makes microRNAs (miRNAs), or Dicer-2, which makes siRNAs, and rasiRNAs lack the 2',3' hydroxy termini characteristic of animal siRNA and miRNA. Unlike siRNAs and miRNAs, rasiRNAs function through the Piwi, rather than the Ago, Argonaute protein subfamily. Our data suggest that rasiRNAs protect the fly germline through a silencing mechanism distinct from both the miRNA and RNA interference pathways.     

Endogenous siRNAs derived from transposons and mRNAs in Drosophila somatic cells

Small interfering RNAs (siRNAs) direct RNA interference (RNAi) in eukaryotes. In flies, somatic cells produce siRNAs from exogenous double-stranded RNA (dsRNA) as a defense against viral infection. We identified endogenous siRNAs (endo-siRNAs), 21 nucleotides in length, that correspond to transposons and heterochromatic sequences in the somatic cells of Drosophila melanogaster. We also detected endo-siRNAs complementary to messenger RNAs (mRNAs); these siRNAs disproportionately mapped to the complementary regions of overlapping mRNAs predicted to form double-stranded RNA in vivo. Normal accumulation of somatic endo-siRNAs requires the siRNA-generating ribonuclease Dicer-2 and the RNAi effector protein Argonaute2 (Ago2). We propose that endo-siRNAs generated by the fly RNAi pathway silence selfish genetic elements in the soma, much as Piwi-interacting RNAs do in the germ line.     

siRNA对乙型脑炎病毒复制的抑制效果

以JEV的NSl基因mRNA为靶序列,设计合成了4个siRNA序列,构建了各自的表达质粒pS-NSlA、pS-NSlB、pS-NSlC和pS-NSlD.通过间接免疫荧光、Western blot检测、RT-PCR和病毒空斑检测等方法对这些siRNAs抑制JEV复制的效果进行了评价.结果表明4个siRNA表达质粒均对JEV在细胞中的复制具有不同程度的抑制作用,其中pS-NSlC、pS-NSlD有显著的抑制作用.说明针对NSl基因的siRNAs可以有效抑制JEV的复制,并有望成为应对JEV感染的治疗方法.     

Methylation as a crucial step in plant microRNA biogenesis

Methylation on the base or the ribose is prevalent in eukaryotic ribosomal RNAs (rRNAs) and is thought to be crucial for ribosome biogenesis and function. Artificially introduced 2'-O-methyl groups in small interfering RNAs (siRNAs) can stabilize siRNAs in serum without affecting their activities in RNA interference in mammalian cells. Here, we show that plant microRNAs (miRNAs) have a naturally occurring methyl group on the ribose of the last nucleotide. Whereas methylation of rRNAs depends on guide RNAs, the methyltransferase protein HEN1 is sufficient to methylate miRNA/miRNA* duplexes. Our studies uncover a new and crucial step in plant miRNA biogenesis and have profound implications in the function of miRNAs.     

Secondary siRNAs result from unprimed RNA synthesis and form a distinct class

In Caenorhabditis elegans, an effective RNA interference (RNAi) response requires the production of secondary short interfering RNAs (siRNAs) by RNA-directed RNA polymerases (RdRPs). We cloned secondary siRNAs from transgenic C. elegans lines expressing a single 22-nucleotide primary siRNA. Several secondary siRNAs start a few nucleotides downstream of the primary siRNA, indicating that non-RISC (RNA-induced silencing complex)-cleaved mRNAs are substrates for secondary siRNA production. In lines expressing primary siRNAs with single-nucleotide mismatches, secondary siRNAs do not carry the mismatch but contain the nucleotide complementary to the mRNA. We infer that RdRPs perform unprimed RNA synthesis. Secondary siRNAs are only of antisense polarity, carry 5' di- or triphosphates, and are only in the minority associated with RDE-1, the RNAi-specific Argonaute protein. Therefore, secondary siRNAs represent a distinct class of small RNAs. Their biogenesis depends on RdRPs, and we propose that each secondary siRNA is an individual RdRP product.     

基于Smad 7基因为靶序列的RNA干涉作用研究

为了探讨小干涉RNA(siRNA)对靶基因Smad7mRNA表达的阻断作用,采用体外转录方法制备Smad7基因的小干涉RNAs(siRNAs),用脂质体介导瞬时转染BERP35T 2肺癌细胞系,并采用Northernblot杂交检测靶基因mRNA的表达丰度。结果表明,根据Smad7编码区序列在体外成功地制备了针对2个不同靶序列的siRNAs;Northernblot杂交显示,在转染siRNA的BERP35T 2细胞中,不管是内源性的还是外源性的,Smad7mRNA的表达丰度均明显下降。说明Smad7基因编码区中542563bp及701722bp2个区域均是siRNA作用的有效靶序列,本研究设计并制备的siRNAs能有效抑制Smad7基因的表达。     

RNA干涉技术及其应用

RNA干涉(RNA interference,RNA1)是由双链RNA导入而引起的转录后基因沉默,它可以作为一种有力的工具在多种有机体中抑制特异性基因的表达。文章简要介绍了RNA干涉的发现史、作用机制、特点及该项技术的用途。RNA1的作用机制可以分为起始阶段和效应阶段。双链RNA被Dicer消化成siRNAs(small interfermg RNAs),进一步形成RNA诱导沉默复合物(RNA-mduced silencmg complex,or RISC),在siRNAs的引导下切割靶mRNA。RNAi技术在疾病的基因治疗、功能基因组学及细胞信号通路分析等力面具有广阔的应用前景。     

Argonaute2 is the catalytic engine of mammalian RNAi

Gene silencing through RNA interference (RNAi) is carried out by RISC, the RNA-induced silencing complex. RISC contains two signature components, small interfering RNAs (siRNAs) and Argonaute family proteins. Here, we show that the multiple Argonaute proteins present in mammals are both biologically and biochemically distinct, with a single mammalian family member, Argonaute2, being responsible for messenger RNA cleavage activity. This protein is essential for mouse development, and cells lacking Argonaute2 are unable to mount an experimental response to siRNAs. Mutations within a cryptic ribonuclease H domain within Argonaute2, as identified by comparison with the structure of an archeal Argonaute protein, inactivate RISC. Thus, our evidence supports a model in which Argonaute contributes "Slicer" activity to RISC, providing the catalytic engine for RNAi.     

A microRNA in a multiple-turnover RNAi enzyme complex

In animals, the double-stranded RNA-specific endonuclease Dicer produces two classes of functionally distinct, tiny RNAs: microRNAs (miRNAs) and small interfering RNAs (siRNAs). miRNAs regulate mRNA translation, whereas siRNAs direct RNA destruction via the RNA interference (RNAi) pathway. Here we show that, in human cell extracts, the miRNA let-7 naturally enters the RNAi pathway, which suggests that only the degree of complementarity between a miRNA and its RNA target determines its function. Human let-7 is a component of a previously identified, miRNA-containing ribonucleoprotein particle, which we show is an RNAi enzyme complex. Each let-7-containing complex directs multiple rounds of RNA cleavage, which explains the remarkable efficiency of the RNAi pathway in human cells.     

VEGFR1基因特异性siRNA的筛选

[目的]筛选血管内皮生长因子受体(VEGRF)基因特异性小干扰RNA(Small Interference RNA,siRNA),为肿瘤等疾病的基因治疗寻找一种新途径。[方法]以高表达VEGFR1的人脐静脉血管内皮细胞(HUVEC)为模型,采用RNA干扰技术,化学合成了3条针对血管内皮生长因子受体1(VEGFR1)的特异性siRNA,用Lipofectamine2000TM转染HUVEC细胞株,通过Real time RT-PCR技术检测HUVEC细胞VEGFR1基因mRNA的表达。并对效果最好的VEGFR1 siRNA-2进行siRNA的浓度梯度效果检测。[结果]结果表明,与对照组相比,所设计的3条siRNA均能不同程度地抑制VEGFR1 mRNA的表达,其中siRNA-2号最有效,浓度为50 nmol/L时抑制率达到95%左右。在浓度梯度试验中,VEGFR1 siRNA-2转染浓度为50 pmol/L时,对VEGFR1基因的沉默效果还能达到50%左右。[结论]所设计的siRNA能有效抑制VEGFR1基因的表达,为RNAi用于靶向VEGFR1的基因治疗提供了非常有效的siRNA序列。     

VEGFR1基因特导性siRNA的筛选

[目的]筛选血管内皮生长因子受体(VEGRF)基因特异性小干扰RNA(Small Interference RNA,siRNA),为肿瘤等疾病的基因治疗寻找一种新途径。[方法]以高表达VEGFR1的人脐静脉血管内皮细胞(HUVEC)为模型,采用RNA干扰技术,化学合成了3条针对血管内皮生长因子受体1(VEGFR1)的特异性siRNA,用Lipofectamine2000TM转染HUVEC细胞株,通过Real time RT-PCR技术检测HUVEC细胞VEGFR1基因mRNA的表达。并对效果最好的VEGFR1 siRNA-2进行siRNA的浓度梯度效果检测。[结果]结果表明,与对照组相比,所设计的3条siRNA均能不同程度地抑制VEGFR1 mRNA的表达,其中siRNA-2号最有效,浓度为50 nmol/L时抑制率达到95%左右。在浓度梯度试验中,VEGFR1 siRNA-2转染浓度为50 pmol/L时,对VEGFR1基因的沉默效果还能达到50%左右。[结论]所设计的siRNA能有效抑制VEGFR1基因的表达,为RNAi用于靶向VEGFR1的基因治疗提供了非常有效的siRNA序列。     

A system for stable expression of short interfering RNAs in mammalian cells

Mammalian genetic approaches to study gene function have been hampered by the lack of tools to generate stable loss-of-function phenotypes efficiently. We report here a new vector system, named pSUPER, which directs the synthesis of small interfering RNAs (siRNAs) in mammalian cells. We show that siRNA expression mediated by this vector causes efficient and specific down-regulation of gene expression, resulting in functional inactivation of the targeted genes. Stable expression of siRNAs using this vector mediates persistent suppression of gene expression, allowing the analysis of loss-of-function phenotypes that develop over longer periods of time. Therefore, the pSUPER vector constitutes a new and powerful system to analyze gene function in a variety of mammalian cell types.     

Role of microRNAs in plant and animal development

Small RNAs, including microRNAs (miRNAs) and short interfering RNAs (siRNAs), are key components of an evolutionarily conserved system of RNA-based gene regulation in eukaryotes. They are involved in many molecular interactions, including defense against viruses and regulation of gene expression during development. miRNAs interfere with expression of messenger RNAs encoding factors that control developmental timing, stem cell maintenance, and other developmental and physiological processes in plants and animals. miRNAs are negative regulators that function as specificity determinants, or guides, within complexes that inhibit protein synthesis (animals) or promote degradation (plants) of mRNA targets.