Cancer proliferation gene discovery through functional genomics

Retroviral short hairpin RNA (shRNA)-mediated genetic screens in mammalian cells are powerful tools for discovering loss-of-function phenotypes. We describe a highly parallel multiplex methodology for screening large pools of shRNAs using half-hairpin barcodes for microarray deconvolution. We carried out dropout screens for shRNAs that affect cell proliferation and viability in cancer cells and normal cells. We identified many shRNAs to be antiproliferative that target core cellular processes, such as the cell cycle and protein translation, in all cells examined. Moreover, we identified genes that are selectively required for proliferation and survival in different cell lines. Our platform enables rapid and cost-effective genome-wide screens to identify cancer proliferation and survival genes for target discovery. Such efforts are complementary to the Cancer Genome Atlas and provide an alternative functional view of cancer cells.     

Systemic RNAi in C. elegans requires the putative transmembrane protein SID-1

Double-stranded RNA-mediated gene interference (RNAi) in Caenorhabditis elegans systemically inhibits gene expression throughout the organism. To investigate how gene-specific silencing information is transmitted between cells, we constructed a strain that permits visualization of systemic RNAi. We used this strain to identify systemic RNA interference-deficient (sid) loci required to spread gene-silencing information between tissues but not to initiate or maintain an RNAi response. One of these loci, sid-1, encodes a conserved protein with predicted transmembrane domains. SID-1 is expressed in cells sensitive to RNAi, is localized to the cell periphery, and is required cell-autonomously for systemic RNAi.     

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.     

逆转录病毒载体RNAi技术抑制猪瘟病毒在猪胚胎成纤维细胞的增殖

利用针对猪瘟病毒(Classical swine fevervirus,CsFV)石门株Npro基因mRNA的shRNA逆转录病毒表达载体,转导猪胚胎成纤维细胞,在G418的筛选压力下,获得7个稳定整合shRNA表达构件的猪胚胎成纤维细胞株.以100TCID50的CSFV分别感染96孔板内的上述细胞克隆,72h后以对感染细胞克隆进行间接免疫荧光分析及子代病毒滴度检测,结果显示,在所获得的7个抗性细胞株中,有3株细胞上猪瘟病毒的增殖显著降低,表明所构建的针对猪瘟病毒Npro基因mRNA的shRNA逆转录病毒整合细胞基因组后转录产生的siRNA可以有效抑制CSFV的复制.     

Transport of dsRNA into cells by the transmembrane protein SID-1

RNA interference (RNAi) spreads systemically in plants and nematodes to silence gene expression distant from the site of initiation. We previously identified a gene, sid-1, essential for systemic but not cell-autonomous RNAi in Caenorhabditis elegans. Here, we demonstrate that SID-1 is a multispan transmembrane protein that sensitizes Drosophila cells to soaking RNAi with a potency that is dependent on double-stranded RNA (dsRNA) length. Further analyses revealed that SID-1 enables passive cellular uptake of dsRNA. These data indicate that systemic RNAi in C. elegans involves SID-1-mediated intercellular transport of dsRNA.     

非洲绿猴肾细胞外源绿色荧光蛋白基因表达的RNA干扰

非洲绿猴肾细胞(Vero)是多种病毒的适应细胞。本研究将外源的绿色荧光蛋白(GFP)基因转染到Veto-E6中,并利用体外转录合成的绿荧光蛋白短双链干扰RNA(siGFP)其表达进行干扰。结果显示：siGFP能有效阻断外源的绿色荧光蛋白基因在Vero-E6细胞中的表达,而不相关的siRNA则对绿荧光蛋白基因的表达没有影响。这为利用RNAi技术在Vero-E6细胞中,抑制外源基因的表达以及通过RNAi技术进行抗病毒的研究奠定了一定的基础。     

人H1启动子转录shRNA的细胞种属特异性研究

利用siDirect软件预测绿色荧光蛋白(GFP)基因特异性小干扰RNA(siRNA),将人工合成的相应shRNA插入含人H1启动子的pSuper载体,获得表达载体pSuper-shRNA,再将H1-shRNA插入表达GFP基因的peGFP-N1载体,获得表达载体peGFP-H1-shRNA。分别以pSuper-shRNA peGFP-N1和peGFP-H1-shRNA转染COS-1、293-T、鸡胚肝(CEL)和鸡胚成纤维(CEF)细胞,根据相同条件下GFP阳性细胞数及荧光强度变化判断产生的siRNA对GFP基因表达的沉默作用,比较人H1启动子在哺乳动物和禽源细胞中的转录活性。结果表明:人H1启动子在2种哺乳动物细胞中能有效转录shRNA,但在2种禽源细胞中的转录活性很弱,提示在禽源细胞中表达siRNA和进行基因沉默研究应选用禽源启动子。     

植物非细胞自主性RNAi及其应用研究进展

非细胞自主性RNAi是发生于那些非应用或非产生dsRNA的细胞或组织中的RNAi,包括系统性RNAi和环境RNAi。系统性RNAi是沉默现象从一个细胞扩散到另一个细胞或从一个组织扩散到另一个组织的过程,环境RNAi是细胞从环境中吸收dsRNA而触发的RNAi。近年来,植物非细胞自主性RNAi研究取得了较大进展,就其机制及应用进行了评述,首次提出在植物中也有环境RNAi存在。     

Genome-wide RNAi analysis of growth and viability in Drosophila cells

A crucial aim upon completion of whole genome sequences is the functional analysis of all predicted genes. We have applied a high-throughput RNA-interference (RNAi) screen of 19,470 double-stranded (ds) RNAs in cultured cells to characterize the function of nearly all (91%) predicted Drosophila genes in cell growth and viability. We found 438 dsRNAs that identified essential genes, among which 80% lacked mutant alleles. A quantitative assay of cell number was applied to identify genes of known and uncharacterized functions. In particular, we demonstrate a role for the homolog of a mammalian acute myeloid leukemia gene (AML1) in cell survival. Such a systematic screen for cell phenotypes, such as cell viability, can thus be effective in characterizing functionally related genes on a genome-wide scale.     

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.     

SMEDWI-2 is a PIWI-like protein that regulates planarian stem cells

We have identified two genes, smedwi-1 and smedwi-2, expressed in the dividing adult stem cells (neoblasts) of the planarian Schmidtea mediterranea. Both genes encode proteins that belong to the Argonaute/PIWI protein family and that share highest homology with those proteins defined by Drosophila PIWI. RNA interference (RNAi) of smedwi-2 blocks regeneration, even though neoblasts are present, irradiation-sensitive, and capable of proliferating in response to wounding; smedwi-2(RNAi) neoblast progeny migrate to sites of cell turnover but, unlike normal cells, fail at replacing aged tissue. We suggest that SMEDWI-2 functions within dividing neoblasts to support the generation of cells that promote regeneration and homeostasis.     

Unexpectedly high levels of HIV-1 RNA and protein synthesis in a cytocidal infection

The expression of a laboratory strain of HIV-1 (HTLV-IIIB) has been studied in mitogen-stimulated peripheral blood lymphocytes (PBLs) and in two lymphoid cell lines (CEM cells and C8166 cells). HIV-expressing cells contained from 300,000 to 2,500,000 copies of viral RNA per cell. Near-synchronous expression of an active infection could be achieved in C8166 cells. In these cells, the high copy numbers of viral RNA used as much as 40% of total protein synthesis for the production of viral gag protein, with high levels of viral RNA and protein synthesis preceding cell death by 2 to 4 days.     

Functional genomic analysis of RNA interference in C. elegans

RNA interference (RNAi) of target genes is triggered by double-stranded RNAs (dsRNAs) processed by conserved nucleases and accessory factors. To identify the genetic components required for RNAi, we performed a genome-wide screen using an engineered RNAi sensor strain of Caenorhabditis elegans. The RNAi screen identified 90 genes. These included Piwi/PAZ proteins, DEAH helicases, RNA binding/processing factors, chromatin-associated factors, DNA recombination proteins, nuclear import/export factors, and 11 known components of the RNAi machinery. We demonstrate that some of these genes are also required for germline and somatic transgene silencing. Moreover, the physical interactions among these potential RNAi factors suggest links to other RNA-dependent gene regulatory pathways.     

RNA interference in Colorado potato beetle(Leptinotarsa decemlineata): A potential strategy for pest control

Colorado potato beetle(CPB), Leptinotarsa decemlineata, is a notorious destructive pest that mainly feeds on the leaves of potato and several other solanaceous plants. CPB is widely recognized for its adaptation to a remarkable variety of host plants and diverse climates, and its high resistance to insecticides and Bacillus thuringiensis toxins. RNA interference(RNAi) is a sequence-specific, endogenous gene silencing mechanism evoked by small RNA molecules that is used as a robust tool for virus and pest control. RNAi has been extensively tested for CPB management by employing various target genes and delivery methods. This article reviews the screening of RNAi target genes, efficient RNAi delivery systems, and factors affecting RNAi efficiency in CPB, which may help understand the mechanisms of RNAi and its application in CPB control strategy.     

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.     

siRNA对鸡传染性支气管炎病毒(IBV)复制的影响

RNA干涉(RNAi)可以通过短双链RNA(siRNA)介导,特异地降解相应的mRNA,从而抑制基因表达,导致转录后水平的基因沉默(PTGS)。本研究利用这种技术,将鸡传染性支气管炎病毒(IBV)感染体外培养的Vero-E6,使其适应Vero-E6,然后用体外转录合成的针对IBV部分基因的siRNA,对其复制进行RNAi。结果显示,针对IBVM蛋白的siRNA能有效地抑制IBV在Vero-E6细胞中的复制。     

Functional genomic analysis of the Wnt-wingless signaling pathway

The Wnt-Wingless (Wg) pathway is one of a core set of evolutionarily conserved signaling pathways that regulates many aspects of metazoan development. Aberrant Wnt signaling has been linked to human disease. In the present study, we used a genomewide RNA interference (RNAi) screen in Drosophila cells to screen for regulators of the Wnt pathway. We identified 238 potential regulators, which include known pathway components, genes with functions not previously linked to this pathway, and genes with no previously assigned functions. Reciprocal-Best-Blast analyses reveal that 50% of the genes identified in the screen have human orthologs, of which approximately 18% are associated with human disease. Functional assays of selected genes from the cell-based screen in Drosophila, mammalian cells, and zebrafish embryos demonstrated that these genes have evolutionarily conserved functions in Wnt signaling. High-throughput RNAi screens in cultured cells, followed by functional analyses in model organisms, prove to be a rapid means of identifying regulators of signaling pathways implicated in development and disease.