An endoribonuclease-prepared siRNA screen in human cells identifies genes essential for cell division

RNA interference (RNAi) is an evolutionarily conserved defence mechanism whereby genes are specifically silenced through degradation of messenger RNAs; this process is mediated by homologous double-stranded (ds)RNA molecules. In invertebrates, long dsRNAs have been used for genome-wide screens and have provided insights into gene functions. Because long dsRNA triggers a nonspecific interferon response in many vertebrates, short interfering (si)RNA or short hairpin (sh)RNAs must be used for these organisms to ensure specific gene silencing. Here we report the generation of a genome-scale library of endoribonuclease-prepared short interfering (esi)RNAs from a sequence-verified complementary DNA collection representing 15,497 human genes. We used 5,305 esiRNAs from this library to screen for genes required for cell division in HeLa cells. Using a primary high-throughput cell viability screen followed by a secondary high content videomicroscopy assay, we identified 37 genes required for cell division. These include several splicing factors for which knockdown generates mitotic spindle defects. In addition, a putative nuclear-export terminator was found to speed up cell proliferation and mitotic progression after knockdown. Thus, our study uncovers new aspects of cell division and establishes esiRNA as a versatile approach for genomic RNAi screens in mammalian cells.     

A conserved siRNA-degrading RNase negatively regulates RNA interference in C. elegans

In many organisms, introducing double-stranded RNA (dsRNA) causes the degradation of messenger RNA that is homologous to the trigger dsRNA--a process known as RNA interference. The dsRNA is cleaved into short interfering RNAs (siRNAs), which hybridize to homologous mRNAs and induce their degradation. dsRNAs vary in their ability to trigger RNA interference: many mRNA-targeting dsRNAs show weak phenotypes, and nearly all mRNAs of the Caenorhabditis elegans nervous system are refractory to RNA interference. C. elegans eri-1 was identified in a genetic screen for mutants with enhanced sensitivity to dsRNAs. Here we show that eri-1 encodes an evolutionarily conserved protein with domains homologous to nucleic-acid-binding and exonuclease proteins. After exposure to dsRNA or siRNAs, animals with eri-1 mutations accumulate more siRNAs than do wild-type animals. C. elegans ERI-1 and its human orthologue degrade siRNAs in vitro. In the nematode worm, ERI-1 is predominantly cytoplasmic and is expressed most highly in the gonad and a subset of neurons, suggesting that ERI-1 siRNase activity suppresses RNA interference more intensely in these tissues. Thus, ERI-1 is a negative regulator that may normally function to limit the duration, cell-type specificity or endogenous functions of RNA interference.     

植物中RNA沉默机制的研究进展

近年来,人们对植物中RNA沉默机制的的认识日渐清晰,小RNAs在其中发挥重要作用.文章综述了植物中RNA沉默的机制、RNA沉默的主要途径及其在防御外源DNA序列入侵过程中的主要功能.并简要介绍了由DNA病毒编码的基因沉默抑制子在对抗宿主沉默反应的作用.文章最后阐述了对RNA沉默进行深入研究的必要性,对需要研究的问题进行了分析,为抗病毒作物育种提供了有力依据.     

植物基因工程中的转基因沉默机制

植物转基因沉默可以发生在染色体DNA、转录和转录后三种不同的层次上,转录水平基因沉默机制涉及DNA甲基化、位置效应、重复序列、同源序列等作用。转录后水平基因沉默机制常用RNA域值模型、异常RNA模型、双链RNA模型、未成熟翻译终止模型等解释。目前没有一种通用的模型可以解释所有的转基因沉默现象,但不同模型从不同方面解释了转基因机制的某些方面。     

RNA干涉与基因沉默研究进展

生物体内导入双链RNA(dsRNA)后会引起体内同源基因特异的沉默，这种现象称为RNA干涉(RNAi)．基因沉默是生物体基因表达调控的一种重要机制，具有重要生物学功能．就RNA干涉与基因沉默的研究历史、作用机制和应用作了综述．     

Prevalence of off-target effects in Drosophila RNA interference screens

RNA interference (RNAi) in both plants and animals is mediated by small RNAs of approximately 21-23 nucleotides in length for regulation of target gene expression at multiple levels through partial sequence complementarities. Combined with widespread genome sequencing, experimental use of RNAi has the potential to interrogate systematically all genes in a given organism with respect to a particular function. However, owing to a tolerance for mismatches and gaps in base-pairing with targets, small RNAs could have up to hundreds of potential target sequences in a genome, and some small RNAs in mammalian systems have been shown to affect the levels of many messenger RNAs besides their intended targets. The use of long double-stranded RNAs (dsRNAs) in Drosophila, where Dicer-mediated processing produces small RNAs inside cells, has been thought to reduce the probability of such 'off-target effects' (OTEs). Here we show, however, that OTEs mediated by short homology stretches within long dsRNAs are prevalent in Drosophila. We have performed a genome-wide RNAi screen for novel components of Wingless (Wg) signal transduction in Drosophila S2R + cells, and found few, if any, legitimate candidates. Rather, many of the top candidates exert their effects on Wg response through OTEs on known pathway components or through promiscuous OTEs produced by tandem trinucleotide repeats present in many dsRNAs and genes. Genes containing such repeats are over-represented in candidate lists from published screens, suggesting that they represent a common class of false positives. Our results suggest simple measures to improve the reliability of genome-wide RNAi screens in Drosophila and other organisms.     

A genetic link between co-suppression and RNA interference in C. elegans

Originally discovered in plants, the phenomenon of co-suppression by transgenic DNA has since been observed in many organisms from fungi to animals: introduction of transgenic copies of a gene results in reduced expression of the transgene as well as the endogenous gene. The effect depends on sequence identity between transgene and endogenous gene. Some cases of co-suppression resemble RNA interference (the experimental silencing of genes by the introduction of double-stranded RNA), as RNA seems to be both an important initiator and a target in these processes. Here we show that co-suppression in Caenorhabditis elegans is also probably mediated by RNA molecules. Both RNA interference and co-suppression have been implicated in the silencing of transposons. We now report that mutants of C. elegans that are defective in transposon silencing and RNA interference (mut-2, mut-7, mut-8 and mut-9) are in addition resistant to co-suppression. This indicates that RNA interference and co-suppression in C. elegans may be mediated at least in part by the same molecular machinery, possibly through RNA-guided degradation of messenger RNA molecules.     

Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs

RNA interference (RNAi) holds considerable promise as a therapeutic approach to silence disease-causing genes, particularly those that encode so-called 'non-druggable' targets that are not amenable to conventional therapeutics such as small molecules, proteins, or monoclonal antibodies. The main obstacle to achieving in vivo gene silencing by RNAi technologies is delivery. Here we show that chemically modified short interfering RNAs (siRNAs) can silence an endogenous gene encoding apolipoprotein B (apoB) after intravenous injection in mice. Administration of chemically modified siRNAs resulted in silencing of the apoB messenger RNA in liver and jejunum, decreased plasma levels of apoB protein, and reduced total cholesterol. We also show that these siRNAs can silence human apoB in a transgenic mouse model. In our in vivo study, the mechanism of action for the siRNAs was proven to occur through RNAi-mediated mRNA degradation, and we determined that cleavage of the apoB mRNA occurred specifically at the predicted site. These findings demonstrate the therapeutic potential of siRNAs for the treatment of disease.     

Differential roles of MDA5 and RIG-I helicases in the recognition of RNA viruses

The innate immune system senses viral infection by recognizing a variety of viral components (including double-stranded (ds)RNA) and triggers antiviral responses. The cytoplasmic helicase proteins RIG-I (retinoic-acid-inducible protein I, also known as Ddx58) and MDA5 (melanoma-differentiation-associated gene 5, also known as Ifih1 or Helicard) have been implicated in viral dsRNA recognition. In vitro studies suggest that both RIG-I and MDA5 detect RNA viruses and polyinosine-polycytidylic acid (poly(I:C)), a synthetic dsRNA analogue. Although a critical role for RIG-I in the recognition of several RNA viruses has been clarified, the functional role of MDA5 and the relationship between these dsRNA detectors in vivo are yet to be determined. Here we use mice deficient in MDA5 (MDA5-/-) to show that MDA5 and RIG-I recognize different types of dsRNAs: MDA5 recognizes poly(I:C), and RIG-I detects in vitro transcribed dsRNAs. RNA viruses are also differentially recognized by RIG-I and MDA5. We find that RIG-I is essential for the production of interferons in response to RNA viruses including paramyxoviruses, influenza virus and Japanese encephalitis virus, whereas MDA5 is critical for picornavirus detection. Furthermore, RIG-I-/- and MDA5-/- mice are highly susceptible to infection with these respective RNA viruses compared to control mice. Together, our data show that RIG-I and MDA5 distinguish different RNA viruses and are critical for host antiviral responses.     

基因沉默抑制子HC-Pro表达载体的构建

RNA沉默广泛存在于植物等大多数真核生物中,能够防御外源基因的入侵和调控基因表达等.然而,基因沉默抑制子HC-Pro蛋白的具体功能的研究并不十分清楚.本文重点介绍了基因沉默抑制子HC-Pro的表达载体pBI121-HC-Pro的构建及HC-Pro基因在烟草(Nicotiana benthamiana)中的稳定表达.并利用半定量RT-PCR技术检测了目的基因HC-Pro的表达,检测结果表明我们获得了HC-Pro基因稳定表达的转基因烟草株系,为进一步深入研究基因沉默抑制子HC-Pro的功能奠定了实验基础.     

RNA干涉及其在肿瘤研究中的应用

RNAi是双链RNA介导的转录后基因沉默的过程，是一种高效的高特异性抑制基因表达的新途径。通过双链小干涉RNA(siRNA)与一系列蛋白质结合形成siRNA诱导的沉默复合体(RISC)并活化，然后，RISC对靶基因进行识别、降解。与反义方法相比，siRNA具有更好的抑制效果。RNAi的应用将为癌症的基因治疗提供新的方法。