Novel Insights Into RNAi Off-Target Effects Using C. Elegans Paralogs (original) (raw)
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Applications of RNAi in C. elegans Research
2008
The discovery of RNA interference in C. elegans in 1998 has established this nematode as a key model to study RNAi mechanisms. Meanwhile, RNAi has been extensively applied to study a variety of basic biological questions in worms. In C. elegans, the dsRNA required to trigger RNAi can be administered by injection, by soaking, and importantly, by feeding. The delivery of dsRNA through food and the existence of two RNAi feeding libraries allow for large scale RNAi approaches. Thus, RNAi libraries have been used to screen for genes related with processes as diverse as embryonic development, longevity, fat accumulation, DNA damage response, axon guidance or synapse structure, among others. Moreover, since RNAi facilitates the inactivation of more than one gene at the time in the same animal, RNAi assays are being applied to uncover genetics interactions among genes and therefore there is a significant and ongoing progress in identifying components of genetics pathways and networks. Here we review successful applications of RNAi as tool in C. elegans research, and discuss trends in using such tool to shed light into several biological research fields. Applications of RNAi mechanisms nuances Since 1998, when Andy Fire and collaborators published in Nature a "Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans" [2], scientists are squeezing all the fresh information published on RNAi mechanisms to get an optimal efficiency and expand the applicability of this revolutionary experimental tool. As examples, C. elegans researchers are now able to conduct RNAi assays in a tissue-specific manner or in strains with hypersensitivity to RNAi. Fire and co-workers observed how injection of dsRNA into worm gonads induced a sequence specific silencing of the corresponding gene. With the help of valuable research in C. elegans, plants and Drosophila, details of RNAi mechanisms have been revealed in the past few years [5, 6]. Once dsRNA is inside the cell, a protein called Dicer slices it to produce small dsRNA molecules of 21-22 nucleotides known as siRNAs. Each siRNA has a "sense" strand (sequence identical to the target gene) that is eliminated and an "antisense" strand (sequence complementary to the target gene) that guides the protein complex RISC to degrade the mRNA transcribed from the targeted gene. RNAi silencing effect was initially thought to happen only through mRNA degradation but two more mechanisms were uncovered acting by blocking transcription [7, 8] and by inhibiting translation [9, 10]. Interestingly, siRNAs are defensive weapons that the eukaryotic cell uses to fight dsRNA viruses and other harmful influences for its genome such as transposons (mobile elements) and repetitive sequences (as transgenes). Thus, worms carrying mutations in genes related with RNAi process are often unable to silence transposons or transgenes in germline tissues [8, 11]. Rather than digging into details of RNAi mechanisms, in this section we intend to list and comment on RNAi mechanism nuances that allow us to profit from RNAi as technique. A remarkable feature of RNAi in C. elegans is the systemic spread of the induced silencing. In other words, application of dsRNA in one part of the animal can produce an RNAi effect in distant tissues. Scientists soon exploited this phenomenon to trigger a systemic RNAi response by soaking worms in dsRNA solution and by feeding worms with bacteria expressing dsRNA [12, 13]. Administration of dsRNA by soaking is more expensive and less reliable than feeding (Fernandez and Piano, personal communication). Moreover, there are two complementary RNAi feeding libraries, commercially available and widely distributed, containing feeding clones to inactivate approximately 86% of the C. elegans genes [14, 15]. These libraries were generated in Julie Ahringer and Marc Vidal labs using as template to synthesize dsRNA genomic DNA and a cDNA library respectively. As consequence, dsRNA by feeding is a technique performed in most of the C. elegans labs routinely. Conveniently RNAi by feeding allows for modulation or titration of the RNAi effect by diluting the bacteria [16, 17]. Interestingly, although the RNA machinery is capable of being saturated, combinatorial RNAi (simultaneous targeting of two different genes) have effectively been developed [16]. The phenomenon of systemic RNAi, or RNAi spreading, has been studied by identifying mutants in which systemic RNAi is impaired [18, 19]. Detailed analysis of these mutations will clarify the spreading process. One of the genes affected for these mutations is sid-1, which encodes a transmembrane protein required for the uptake of dsRNA. In the absence of SID-1 protein, dsRNA can still trigger RNAi in cells where is injected or endogenously synthesized, but it is not effective in other neighboring or distant cells.
RNAI: GENE SILENCING APPROACH IN C. elegans and H. sapiens
International Journal of Molecular Biology, 2010
siRNAs (Short interfering RNAs) and miRNAs (microRNAs), which are mediate silencing via distinct mechanisms C. elegans and Homo sapiens. The breaking of a doublestranded RNA (dsRNA) matching a specific gene sequence into short pieces called short interfering RNA, which trigger the degradation of mRNA that matches its sequence. In this review, we discussed the RNA interference with its principle and application in recent biological research areas. Principal The phenomenon of RNAi, originally described in the nematode worm C. elegans by Fire and colleagues in 1998, has been recognized as a general mechanism in many organisms. Basically, RNAi is induced within the cytoplasm when long, double-stranded RNA (dsRNA) is recognized by Dicer, a multidomain RNase III enzyme. Dicer processes dsRNA into short (21-25 nucleotides) duplexes that are termed siRNAs. Like products of other RNase III enzymes, siRNA duplexes contain 5′ phosphate and 3′ hydroxyl termini, and two single-stranded nucleotide overhangs on their 3′ ends. These structural features are important for the entry of siRNAs into the RNAi pathway because blunt-ended siRNAs or those that lack a 5′ phosphate group are ineffective in triggering gene silencing [41]. It is processed by the RNase-III enzyme Dicer into 21-28nucleotide double-stranded siRNA duplexes. The RISC associated enzyme Slicer (Argonaute-2) degrades the target mRNA.
In C. elegans, High Levels of dsRNA Allow RNAi in the Absence of RDE-4
PLoS ONE, 2008
C. elegans Dicer requires an accessory double-stranded RNA binding protein, RDE-4, to enact the first step of RNA interference, the cleavage of dsRNA to produce siRNA. While RDE-4 is typically essential for RNAi, we report that in the presence of high concentrations of trigger dsRNA, rde-4 deficient animals are capable of silencing a transgene. By multiple criteria the silencing occurs by the canonical RNAi pathway. For example, silencing is RDE-1 dependent and exhibits a decrease in the targeted mRNA in response to an increase in siRNA. We also find that high concentrations of dsRNA trigger lead to increased accumulation of primary siRNAs, consistent with the existence of a rate-limiting step during the conversion of primary to secondary siRNAs. Our studies also revealed that transgene silencing occurs at low levels in the soma, even in the presence of ADARs, and that at least some siRNAs accumulate in a temperature-dependent manner. We conclude that an RNAi response varies with different conditions, and this may allow an organism to tailor a response to specific environmental signals.
Functional Genomic Analysis of RNA Interference in C. elegans
Science, 2005
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 RNAdependent gene regulatory pathways.
Multiple small RNA pathways regulate the silencing of repeated and foreign genes in C. elegans
Genes & Development, 2013
Gene segments from other organisms, such as viruses, are detected as foreign and targeted for silencing by RNAi pathways. A deep-sequencing map of the small RNA response to repeated transgenes introduced to Caenorhabditis elegans revealed that specific segments are targeted by siRNAs. Silencing of the foreign gene segments depends on an antiviral response that involves changes in active and silent chromatin modifications and altered levels of antisense siRNAs. Distinct Argonaute proteins target foreign genes for silencing or protection against silencing. We used a repeated transgene in a genome-wide screen to identify gene disruptions that enhance silencing of foreign genetic elements and identified 69 genes. These genes cluster in four groups based on overlapping sets of coexpressed genes, including a group of germline-expressed genes that are likely coregulated by the E2F transcription factor. Many of the gene inactivations enhance exogenous RNAi. About half of the 69 genes have r...
Background: The approach of RNAi mediated gene knockdown, employing exogenous dsRNA, is being beneficially exploited in various fields of functional genomics. The immense utility of the approach came to fore from studies with model system C. elegans, but quickly became applicable with varied research models ranging from in vitro to various in vivo systems. Previously, there have been reports on the refractoriness of the neuronal cells to RNAi mediated gene silencing following which several modulators like eri-1 and lin-15 were described in C. elegans which, when present, would negatively impact the gene knockdown.
Editorial Novapublishers CHAPTER TITLE : Applications of RNAi in C . elegans research
2007
The discovery of RNA interference in C. elegans in 1998 has established this nematode as a key model to study RNAi mechanisms. Meanwhile, RNAi has been extensively applied to study a variety of basic biological questions in worms. In C. elegans, the dsRNA required to trigger RNAi can be administered by injection, by soaking, and importantly, by feeding. The delivery of dsRNA through food and the existence of two RNAi feeding libraries allow for large scale RNAi approaches. Thus, RNAi libraries have been used to screen for genes related with processes as diverse as embryonic development, longevity, fat accumulation, DNA damage response, axon guidance or synapse structure, among others. Moreover, since RNAi facilitates the inactivation of more than one gene at the time in the same animal, RNAi assays are being applied to uncover genetics interactions among genes and therefore there is a significant and ongoing progress in identifying components of genetics pathways and networks. Here ...
Cell, 2006
Argonaute (AGO) proteins interact with small RNAs to mediate gene silencing. C. elegans contains 27 AGO genes, raising the question of what roles these genes play in RNAi and related gene-silencing pathways. Here we describe 31 deletion alleles representing all of the previously uncharacterized AGO genes. Analysis of single-and multiple-AGO mutant strains reveals functions in several pathways, including (1) chromosome segregation, (2) fertility, and (3) at least two separate steps in the RNAi pathway. We show that RDE-1 interacts with trigger-derived sense and antisense RNAs to initiate RNAi, while several other AGO proteins interact with amplified siRNAs to mediate downstream silencing. Overexpression of downstream AGOs enhances silencing, suggesting that these proteins are limiting for RNAi. Interestingly, these AGO proteins lack key residues required for mRNA cleavage. Our findings support a two-step model for RNAi, in which functionally and structurally distinct AGOs act sequentially to direct gene silencing.
Natural and Unanticipated Modifiers of RNAi Activity in Caenorhabditis elegans
PLoS ONE, 2012
Organisms used as model genomics systems are maintained as isogenic strains, yet evidence of sequence differences between independently maintained wild-type stocks has been substantiated by whole-genome resequencing data and strain-specific phenotypes. Sequence differences may arise from replication errors, transposon mobilization, meiotic gene conversion, or environmental or chemical assault on the genome. Low frequency alleles or mutations with modest effects on phenotypes can contribute to natural variation, and it has proven possible for such sequences to become fixed by adapted evolutionary enrichment and identified by resequencing. Our objective was to identify and analyze single locus genetic defects leading to RNAi resistance in isogenic strains of Caenorhabditis elegans. In so doing, we uncovered a mutation that arose de novo in an existing strain, which initially frustrated our phenotypic analysis. We also report experimental, environmental, and genetic conditions that can complicate phenotypic analysis of RNAi pathway defects. These observations highlight the potential for unanticipated mutations, coupled with genetic and environmental phenomena, to enhance or suppress the effects of known mutations and cause variation between wild-type strains.
RNAi pathway integration in Caenorhabditis elegans development
Functional & Integrative Genomics
In this review, the pathways involving small RNAs are provided followed by a new and updated network that illustrates their interplay with diverse cellular mechanisms in Caenorhabditis elegans. The RNA silencing pathways are now recognized as key factors that connect together the many variations in biological processes, including transcriptional gene regulation, post-transcriptional gene silencing, translational gene silencing, apoptosis, meiosis, and antiviral defense. The utilization of small RNAs represents a specific, energy conserving, and fast mechanism of gene regulation via a core system known as RNA interference.