Gene silencing in Caenorhabditis elegans by transitive RNA interference

When a cell is exposed to double-stranded RNA (dsRNA), mRNA from the homologous gene is selectively degraded by a process called RNA interference (RNAi). Here, we provide evidence that dsRNA is amplified in Caenorhabditis elegans to ensure a robust RNAi response. Our data suggest a model in which mRNA targeted by RNAi functions as a template for 5' to 3' synthesis of new dsRNA (termed transitive RNAi). Strikingly, the effect is nonautonomous: dsRNA targeted to a gene expressed in one cell type can lead to transitive RNAi-mediated silencing of a second gene expressed in a distinct cell type. These data suggest dsRNA synthesized in vivo can mediate systemic RNAi.     

Genes required for systemic RNA interference in Caenorhabditis elegans

RNA interference (RNAi) in the nematode worm, Caenorhabditis elegans, occurs systemically. Double-stranded RNA (dsRNA) provided in the diet can be absorbed from the gut lumen and distributed throughout the body, triggering RNAi in tissues that are not exposed to the initial dsRNA trigger. This is in marked contrast to other animals, in which RNAi does not spread from targeted tissues to neighboring cells. Here, we report the characterization of mutants defective in the systemic aspect of RNAi, but not in the core RNAi process itself. Analysis of these mutants suggests that dsRNA uptake is a specific process involving several unique proteins.     

Tissue specificity of Caenorhabditis elegans enhanced RNA interference mutants

Gene knockdown by RNA interference (RNAi) in Caenorhabditis elegans is readily achieved by feeding bacteria expressing double-stranded RNA (dsRNA). Enhanced RNAi (Eri) mutants facilitate RNAi due to their hypersensitivity to dsRNA. Here, we compare eight Eri mutants for sensitivity to ingested dsRNA, targeting a variety of tissue-specific genes.     

A Caenorhabditis elegans RNA-Directed RNA Polymerase in Sperm Development and Endogenous RNA Interference

Short interfering RNAs (siRNAs) are a class of regulatory effectors that enforce gene silencing through formation of RNA duplexes. Although progress has been made in identifying the capabilities of siRNAs in silencing foreign RNA and transposable elements, siRNA functions in endogenous gene regulation have remained mysterious. In certain organisms, siRNA biosynthesis involves novel enzymes that act as RNA-directed RNA polymerases (RdRPs). Here we analyze the function of a Caenorhabditis elegans RdRP, RRF-3, during spermatogenesis. We found that loss of RRF-3 function resulted in pleiotropic defects in sperm development and that sperm defects led to embryonic lethality. Notably, sperm nuclei in mutants of either rrf-3 or another component of the siRNA pathway, eri-1, were frequently surrounded by ectopic microtubule structures, with spindle abnormalities in a subset of the resulting embryos. Through high-throughput small RNA sequencing, we identified a population of cellular mRNAs from spermatogenic cells that appear to serve as templates for antisense siRNA synthesis. This set of genes includes the majority of genes known to have enriched expression during spermatogenesis, as well as many genes not previously known to be expressed during spermatogenesis. In a subset of these genes, we found that RRF-3 was required for effective siRNA accumulation. These and other data suggest a working model in which a major role of the RRF-3/ERI pathway is to generate siRNAs that set patterns of gene expression through feedback repression of a set of critical targets during spermatogenesis.REPRESSION of gene expression by small RNAs of ∼20–30 nt in length is important for many aspects of multicellular eukaryotic development. A variety of classes of small RNA with distinct structural features, modes of biogenesis, and biological functions have been identified (reviewed in Hutvagner and Simard 2008). We are particularly interested in a class of small RNAs, called endogenous short interfering RNAs (siRNAs), that are similar to intermediates in exogenously triggered RNA interference (RNAi) in their perfect complementarity to mRNA targets. High-throughput sequencing technology has provided a valuable tool for characterization of endogenous siRNA populations from many diverse sources, including mouse embryonic stem cells (Babiarz et al. 2008), Drosophila heads (Ghildiyal et al. 2008), and Arabidopsis pollen (Slotkin et al. 2009). These siRNAs have been proposed to function in the regulation of both cellular processes and genome defense through downregulation of gene expression. Caenorhabditis elegans, like plants and fungi, utilizes RNA-copying enzymes called RNA-directed RNA polymerases (RdRPs) as part of the RNAi machinery (Smardon et al. 2000; Sijen et al. 2001). While two of the C. elegans RdRPs are nonessential (RRF-1 and RRF-2), mutations in either of the remaining two (EGO-1 or RRF-3) lead to fertility defects (Smardon et al. 2000; Simmer et al. 2002). RRF-3 is functionally distinct from EGO-1 in that the RRF-3 requirement in fertility is temperature dependent. In addition, RRF-3 activity has an inhibitory effect on exogenously triggered RNAi (resulting in an ERI, or enhanced RNAi, mutant phenotype in rrf-3 mutants). Mutants lacking either RRF-3 or another ERI factor, ERI-1, have been used as experimental tools because of their enhanced sensitivity in RNAi-based screens. One proposed mechanism for the enhancement in RNAi in rrf-3 and eri mutants has been a competition for cofactors between the exogenously triggered RNAi pathway and an endogenous RNAi pathway. Consistent with this hypothesis, siRNAs corresponding to several genes have been shown by Northern analysis to depend upon RRF-3 and other ERI factors for their accumulation (Duchaine et al. 2006; Lee et al. 2006; Yigit et al. 2006). Global microarray analyses have also been undertaken to identify messenger RNAs whose expression is affected by RRF-3 and ERI-1 (Lee et al. 2006; Asikainen et al. 2007).A functional significance of the RRF-3/ERI pathway has been inferred by the inability of rrf-3, eri-1, eri-3, and eri-5 mutant strains to propagate at a high growth temperature (Simmer et al. 2002; Duchaine et al. 2006). Rather than producing temperature-sensitive mutant protein effects, RRF-3 and other ERI proteins are thought to act in a temperature-sensitive process, as evidenced by the predicted truncated and presumed nonfunctional protein fragments that would result from the available deletion alleles and by their shared temperature-sensitive phenotypes. rrf-3 mutant animals have been observed to exhibit X-chromosome missegregation (Simmer et al. 2002) and an unusual persistence of a chromatin mark on the X chromosome during male spermatogenesis (Maine et al. 2005). X-chromosome missegregation and defective spermatogenesis have been referred to in previous studies of eri-1 (Kennedy et al. 2004) and eri-3 and eri-5 (Duchaine et al. 2006). Furthermore, eri-3 mutant sterility can be rescued by insemination with wild-type sperm (Duchaine et al. 2006).Here we investigated the role of RRF-3 during spermatogenesis. We found defects evident at multiple stages, including after fertilization, where defects in rrf-3 mutant sperm can produce subsequent nonviable embryos. By using high-throughput sequencing, we characterized a large population of siRNAs present in spermatogenic cells and found a strong enrichment for antisense siRNAs from genes with known mRNA expression during spermatogenesis. While the majority of siRNA production during spermatogenesis does not require RRF-3, we found a set of genes for which siRNA production was dependent upon RRF-3. Existing data indicate increased expression for these genes in rrf-3 and/or eri-1 mutants. Taken together, our analyses suggest a working model in which the RRF-3/ERI pathway generates siRNAs that downregulate specific genes during spermatogenesis, with this regulation playing a key role in generating functional sperm.     

Effectiveness of specific RNA-mediated interference through ingested double-stranded RNA in Caenorhabditis elegans

Background

In Caenorhabditis elegans, injection of double-stranded RNA (dsRNA) results in the specific inactivation of genes containing homologous sequences, a technique termed RNA-mediated interference (RNAi). It has previously been shown that RNAi can also be achieved by feeding worms Escherichia coli expressing dsRNA corresponding to a specific gene; this mode of dsRNA introduction is conventionally considered to be less efficient than direct injection, however, and has therefore seen limited use, even though it is considerably less labor-intensive.

Results

Here we present an optimized feeding method that results in phenotypes at least as strong as those produced by direct injection of dsRNA for embryonic lethal genes, and stronger for genes with post-embryonic phenotypes. In addition, the interference effect generated by feeding can be titrated to uncover a series of hypomorphic phenotypes informative about the functions of a given gene. Using this method, we screened 86 random genes on consecutive cosmids and identified functions for 13 new genes. These included two genes producing an uncoordinated phenotype (a previously uncharacterized POU homeodomain gene, ceh-6, and a gene encoding a MADS-box protein) and one gene encoding a novel protein that results in a high-incidence-of-males phenotype.

Conclusions

RNAi by feeding can provide significant information about the functions of an individual gene beyond that provided by injection. Moreover, it can be used for special applications for which injection or the use of mutants is sometimes impracticable (for example, titration, biochemistry and large-scale screening). Thus, RNAi by feeding should make possible new experimental approaches for the use of genomic sequence information.     

RNA interference in Caenorhabditis elegans: uptake, mechanism, and regulation

RNA interference (RNAi) is a powerful research tool that has enabled molecular insights into gene activity, pathway analysis, partial loss-of-function phenotypes, and large-scale genomic discovery of gene function. While RNAi works extremely well in the non-parasitic nematode C. elegans, it is also especially useful in organisms that lack facile genetic analysis. Extensive genetic analysis of the mechanisms, delivery and regulation of RNAi in C. elegans has provided mechanistic and phenomenological insights into why RNAi is so effective in this species. These insights are useful for the testing and development of RNAi in other nematodes, including parasitic nematodes where more effective RNAi would be extremely useful. Here, we review the current advances in C. elegans for RNA delivery methods, regulation of cell autonomous and systemic RNAi phenomena, and implications of enhanced RNAi mutants. These discussions, with a focus on mechanism and cross-species application, provide new perspectives for optimizing RNAi in other species.     

Quantitative high-throughput analysis of synthetic genetic interactions in Caenorhabditis elegans by RNA interference

Biological processes are highly dynamic but the current representation of molecular networks is static and largely qualitative. To investigate the dynamic property of genetic networks, a novel quantitative high-throughput method based on RNA interference and capable of calculating the relevance of each interaction, was developed. With this approach, it will be possible to identify not only the components of a network, but also to investigate quantitatively how network and biological processes react to perturbations. As a first application of this method, the genetic interactions of a weak loss-of-function mutation in the gene efl-1/E2F with all the genes of chromosome III were investigated during embryonic development of Caenorhabditis elegans. Fifteen synthetic genetic interactions of efl-1/E2F with the genes of chromosome III were detected, measured and ranked by statistical relevance.     

RDE-2 interacts with MUT-7 to mediate RNA interference in Caenorhabditis elegans

In Caenorhabditis elegans, the activity of transposable elements is repressed in the germline. One of the mechanisms involved in this repression is RNA interference (RNAi), a process in which dsRNA targets cleavage of mRNAs in a sequence-specific manner. The first gene found to be involved in RNAi and transposon silencing in C.elegans is mut-7, a gene encoding a putative exoribonuclease. Here, we show that the MUT-7 protein resides in complexes of ~250 kDa in the nucleus and in the cytosol. In addition, we find that upon triggering of RNAi the cytosolic MUT-7 complex increases in size. This increase is independent of the presence of target RNA, but does depend on the presence of RDE-1 and RDE-4, two proteins involved in small interfering RNA (siRNA) production. Finally, using a yeast two-hybrid screen, we identified RDE-2/MUT-8 as one of the other components of this complex. This protein is encoded by the rde-2/mut-8 locus, previously implicated in RNAi and transposon silencing. Using genetic complementation analysis, we show that the interaction between these two proteins is required for efficient RNAi in vivo. Together these data support a role for the MUT-7/RDE-2 complex downstream of siRNA formation, but upstream of siRNA mediated target RNA recognition, possibly indicating a role in the siRNA amplification step.     

Reverse genetic analysis of the Caenorhabditis elegans 26S proteasome subunits by RNA interference

Reverse genetic analysis was performed on the Caenorhabditis elegans 26S proteasome subunit genes by double-stranded RNA-mediated interference (RNAi). Embryonic and post-embryonic lethality was caused by interference of all of the eight tested 20S core subunits and all of the 19S regulatory particle subunits except for Ce-Rpn9, Ce-Rpn10, and Ce-Rpn12, where RNAi caused no abnormality. However, synthetic suppression of Ce-Rpn10 and Ce-Rpn12 was lethal, whereas neither the combination of Ce-Rpn9 with Ce-Rpn10 nor with Ce-Rpn12 resulted in abnormalities in RNAi. These results indicate that the 26S proteasome is indispensable for embryogenesis and post-embryonic development, although Ce-Rpn9, Ce-Rpn10, and Ce-Rpn12 are not essential, at least under the conditions used. Ce-Rpn10 and Ce-Rpn12 are considered to compensate for the suppression of each other.     

Effective RNA interference in cultured silkworm cells mediated by overexpression of Caenorhabditis elegans SID-1

RNA interference (RNAi) is a conserved mechanism that catalyzes sequence-specific gene silencing and has been used for loss-of-function genetic screens in many organisms. Here, we demonstrated that the expression of Caenorhabditis elegans SID-1 (CeSID-1) could trigger effective gene silencing in the cultured silkworm cell line, BmN4 (BmN4-SID1). Soaking the BmN4-SID1 in dsRNA corresponding to endogenous target genes induced a significant decrease of the amount of mRNA or protein. A small amount of dsRNA was enough to silence the target gene in a few days. Overexpression of CeSID-1 did not affect the cell viability. Our results suggest that BmN4-SID1 can be used in many applications in silkworm cells and will become a valuable resource for gene analysis.     

Inducible systemic RNA silencing in Caenorhabditis elegans

Introduction of double-stranded RNA (dsRNA) can elicit a gene-specific RNA interference response in a variety of organisms and cell types. In many cases, this response has a systemic character in that silencing of gene expression is observed in cells distal from the site of dsRNA delivery. The molecular mechanisms underlying the mobile nature of RNA silencing are unknown. For example, although cellular entry of dsRNA is possible, cellular exit of dsRNA from normal animal cells has not been directly observed. We provide evidence that transgenic strains of Caenorhabditis elegans transcribing dsRNA from a tissue-specific promoter do not exhibit comprehensive systemic RNA interference phenotypes. In these same animals, modifications of environmental conditions can result in more robust systemic RNA silencing. Additionally, we find that genetic mutations can influence the systemic character of RNA silencing in C. elegans and can separate mechanisms underlying systemic RNA silencing into tissue-specific components. These data suggest that trafficking of RNA silencing signals in C. elegans is regulated by specific physiological and genetic factors.     

Distinct roles for RDE-1 and RDE-4 during RNA interference in Caenorhabditis elegans.

RNA interference (RNAi) is a cellular defense mechanism that uses double-stranded RNA (dsRNA) as a sequence-specific trigger to guide the degradation of homologous single-stranded RNAs. RNAi is a multistep process involving several proteins and at least one type of RNA intermediate, a population of small 21-25 nt RNAs (called siRNAs) that are initially derived from cleavage of the dsRNA trigger. Genetic screens in Caenorhabditis elegans have identified numerous mutations that cause partial or complete loss of RNAi. In this work, we analyzed cleavage of injected dsRNA to produce the initial siRNA population in animals mutant for rde-1 and rde-4, two genes that are essential for RNAi but that are not required for organismal viability or fertility. Our results suggest distinct roles for RDE-1 and RDE-4 in the interference process. Although null mutants lacking rde-1 show no phenotypic response to dsRNA, the amount of siRNAs generated from an injected dsRNA trigger was comparable to that of wild-type. By contrast, mutations in rde-4 substantially reduced the population of siRNAs derived from an injected dsRNA trigger. Injection of chemically synthesized 24- or 25-nt siRNAs could circumvent RNAi resistance in rde-4 mutants, whereas no bypass was observed in rde-1 mutants. These results support a model in which RDE-4 is involved before or during production of siRNAs, whereas RDE-1 acts after the siRNAs have been formed.     

细菌介导的发夹结构双链RNA对秀丽小杆线虫中par-1基因的RNA干扰

秀丽小杆线虫(Caenorhabditis elegans)是一种重要的模式生物,目前在C.elegans中发现的许多基因在序列和功能上与哺乳动物的基因有很高的同源性,对其基因功能的研究有助于阐明哺乳动物的基因功能。为检测细菌介导的通过发夹结构表达双链RNA(dsRNA)的RNA干扰(RNAi)在C.elegans中的作用效果,我们设计并构建了可以转录表达母源极性基因par-1的发夹dsRNA的RNAi载体,并转入E.coli HT115中,经过IPTG诱导后浓缩。浓缩菌液在25℃下喂食C.elegans48h后,观察C.elegans胚胎发育情况,同时以含有空载体的菌液作为对照,并与par-1突变体及野生型比较。结果显示:发夹结构表达的dsRNA对par-1基因进行RNAi后,虫体内par-1 mRNA几乎消失,C.elegans早期胚胎分裂不对称性丧失,par-1(RNAi)干扰率在95%以上。     

pWormgatePro enables promoter-driven knockdown by hairpin RNA interference of muscle and neuronal gene products in Caenorhabditis elegans

Recent advances in genome research and RNA interference (RNAi) technology have accelerated the adoption of genome-wide experimental approaches for determining gene function in the model organism Caenorhabditis elegans. Despite recent successes, the application of RNAi is limited when gene knockdown causes complex phenotypes or embryonic lethality. Recently, the high-throughput pWormgate cloning system has been introduced as a tool to efficiently generate heat-shock-inducible hairpin RNA constructs using the Gateway recombination technology. We have modified pWormgate into a versatile hairpin cloning plasmid, pWormgatePro, which facilitates temporally and spatially inducible hairpin RNAi using constitutively active, tissue-specific promoters. To demonstrate its utility we knocked down unc-22 in body wall muscles as well as the axon guidance gene unc-5 in the nervous system indicating that promoter-driven hairpins can overcome the neuronal resistance to RNAi. Using pWormgatePro we also show that RNAi in the nervous system of C. elegans is non-autonomous and that spreading of the RNAi signal from neurons to muscle is substantially reduced but not abolished in spreading-defective sid-1 mutant animals. Our findings illustrate the effectiveness of pWormgatePro for gene silencing in muscle cells and neurons and bring forward the possibility of applying tissue-specific RNAi on a genome-wide scale.