SID-4/NCK-1 is important for dsRNA import in Caenorhabditis elegans.

RNA interference is sequence-specific gene silencing triggered by double-stranded RNA. Systemic RNA interference is where double-stranded RNA, expressed or introduced into 1 cell, is transported to and initiates RNA interference in other cells. Systemic RNA interference is very efficient in Caenorhabditis elegans and genetic screens for systemic RNA interference-defective mutants have identified RNA transporters (SID-1, SID-2, and SID-5) and a signaling protein (SID-3). Here, we report that SID-4 is nck-1, a C. elegans NCK-like adaptor protein. sid-4 null mutations cause a weak, dose-sensitive, systemic RNA interference defect and can be effectively rescued by SID-4 expression in target tissues only, implying a role in double-stranded RNA import. SID-4 and SID-3 (ACK-1 kinase) homologs interact in mammals and insects, suggesting that they may function in a common signaling pathway; however, a sid-3; sid-4 double mutants showed additive resistance to RNA interference, suggesting that these proteins likely interact with other signaling pathways as well. A bioinformatic screen coupled to RNA interference sensitivity tests identified 23 additional signaling components with weak RNA interference-defective phenotypes. These observations suggest that environmental conditions may modulate systemic RNA interference efficacy, and indeed, sid-3 and sid-4 are required for growth temperature effects on systemic RNA interference silencing efficiency.

Efficient Homologous Recombination in Mice Using Long Single Stranded DNA and CRISPR Cas9 Nickase.

The CRISPR/Cas9 nickase mutant is less prone to off-target double-strand (ds)DNA breaks than wild-type Cas9 because to produce dsDNA cleavage it requires two guide RNAs to target the nickase to nearby opposing strands. Like wild-type Cas9 lesions, these staggered lesions are repaired by either non-homologous end joining or, if a repair template is provided, by homologous recombination (HR). Here, we report very efficient (up to 100%) recovery of heterozygous insertions in Mus musculus produced by long (>300 nt), single-stranded DNA donor template-guided repair of paired-nickase lesions.

Intergenerational Transmission of Gene Regulatory Information in Caenorhabditis elegans.

Epigenetic mechanisms can stably maintain gene expression states even after the initiating conditions have changed. Often epigenetic information is transmitted only to daughter cells, but evidence is emerging, in both vertebrate and invertebrate systems, for transgenerational epigenetic inheritance (TEI), the transmission of epigenetic gene regulatory information across generations. Each new description of TEI helps uncover the properties, molecular mechanisms and biological roles for TEI. The nematode Caenorhabditis elegans has been particularly instrumental in the effort to understand TEI, as multiple environmental and genetic triggers can initiate an epigenetic signal that can alter the expression of both transgenes and endogenous loci. Here, we review recent studies of TEI in C. elegans.

SID-1 Domains Important for dsRNA Import in Caenorhabditis elegans.

In the nematode Caenorhabditis elegans, RNA interference (RNAi) triggered by double-stranded RNA (dsRNA) spreads systemically to cause gene silencing throughout the organism and its progeny. We confirm that Caenorhabditis nematode SID-1 orthologs have dsRNA transport activity and demonstrate that the SID-1 paralog CHUP-1 does not transport dsRNA. Sequence comparison of these similar proteins, in conjunction with analysis of loss-of-function missense alleles, identifies several conserved 2-7 amino acid microdomains within the extracellular domain (ECD) that are important for dsRNA transport. Among these missense alleles, we identify and characterize a sid-1 allele, qt95, which causes tissue-specific silencing defects most easily explained as a systemic RNAi export defect. However, we conclude from genetic and biochemical analyses that sid-1(qt95) disrupts only import, and speculate that the apparent export defect is caused by the cumulative effect of sequentially impaired dsRNA import steps. Thus, consistent with previous studies, we fail to detect a requirement for sid-1 in dsRNA export, but demonstrate for the first time that SID-1 functions in the intestine to support environmental RNAi (eRNAi).

SIDT2 Transports Extracellular dsRNA into the Cytoplasm for Innate Immune Recognition.

Double-stranded RNA (dsRNA) is a common by-product of viral infections and acts as a potent trigger of antiviral immunity. In the nematode C. elegans, sid-1 encodes a dsRNA transporter that is highly conserved throughout animal evolution, but the physiological role of SID-1 and its orthologs remains unclear. Here, we show that the mammalian SID-1 ortholog, SIDT2, is required to transport internalized extracellular dsRNA from endocytic compartments into the cytoplasm for immune activation. Sidt2-deficient mice exposed to extracellular dsRNA, encephalomyocarditis virus (EMCV), and herpes simplex virus 1 (HSV-1) show impaired production of antiviral cytokines and-in the case of EMCV and HSV-1-reduced survival. Thus, SIDT2 has retained the dsRNA transport activity of its C. elegans ortholog, and this transport is important for antiviral immunity.

SID-1 Functions in Multiple Roles To Support Parental RNAi in Caenorhabditis elegans.

Systemic RNA interference (RNAi) in Caenorhbaditis elegans requires sid-1, sid-3, and sid-5 Injected, expressed, or ingested double-stranded RNA (dsRNA) is transported between cells, enabling RNAi in most tissues, including the germline and progeny (parental RNAi). A recent report claims that parental RNAi also requires the yolk receptor rme-2 Here, we characterize the role of the sid genes and rme-2 in parental RNAi. We identify multiple independent paths for maternal dsRNA to reach embryos and initiate RNAi. We showed previously that maternal and embryonic sid-1 contribute independently to parental RNAi. Here we demonstrate a role for embryonic sid-5, but not sid-2 or sid-3 in parental RNAi. We also find that maternal rme-2 contributes to but is not required for parental RNAi. We determine that parental RNAi by feeding occurs nearly exclusively in adults. We also introduce 5-ethynyluridine to densely internally label dsRNA, avoiding complications associated with other labeling strategies such as inhibition of normal dsRNA trafficking and separation of label and RNA. Labeling shows that yolk and dsRNA do not colocalize following endocytosis, suggesting independent uptake, and, furthermore, dsRNA appears to rapidly progress through the RAB-7 endocytosis pathway independently of sid-1 activity. Our results support the premise that although sid-1 functions in multiple roles, it alone is central and absolutely required for inheritance of silencing RNAs.

Early Developmental Exposure to dsRNA Is Critical for Initiating Efficient Nuclear RNAi in C. elegans.

RNAi has enabled researchers to study the function of many genes. However, it is not understood why some RNAi experiments succeed while others do not. Here, we show in C. elegans that pharyngeal muscle is resistant to RNAi when initially exposed to double-stranded RNA (dsRNA) by feeding but sensitive to RNAi in the next generation. Investigating this observation, we find that pharyngeal muscle cells as well as vulval muscle cells require nuclear rather than cytoplasmic RNAi. Further, we find in these cell types that nuclear RNAi silencing is most efficiently triggered during early development, defining a critical period for initiating nuclear RNAi. Finally, using heat-shock-induced dsRNA expression, we show that synMuv B class mutants act in part to extend this critical window. The synMuv-B-dependent early-development-associated critical period for initiating nuclear RNAi suggests that mechanisms that restrict developmental plasticity may also restrict the initiation of nuclear RNAi.

Stable Heritable Germline Silencing Directs Somatic Silencing at an Endogenous Locus.

The importance of transgenerationally inherited epigenetic states to organismal fitness remains unknown as well-documented examples are often not amenable to mechanistic analysis or rely on artificial reporter loci. Here we describe an induced silenced state at an endogenous locus that persists, at 100% transmission without selection, for up to 13 generations. This unusually persistent silencing enables a detailed molecular genetic analysis of an inherited epigenetic state. We find that silencing is dependent on germline nuclear RNAi factors and post-transcriptional mechanisms. Consistent with these later observations, inheritance does not require the silenced locus, and we provide genetic evidence that small RNAs embody the inherited silencing signal. Notably, heritable germline silencing directs somatic epigenetic silencing. Somatic silencing does not require somatic nuclear RNAi but instead requires both maternal germline nuclear RNAi and chromatin-modifying activity. Coupling inherited germline silencing to somatic silencing may enable selection for physiologically important traits.

The RNA binding protein MEX-3 retains asymmetric activity in the early Caenorhabditis elegans embryo in the absence of asymmetric protein localization.

The RNA binding protein MEX-3 is required to restrict translation of pal-1, the Caenorhabditis elegans caudal homolog, to the posterior of the early embryo. MEX-3 is present uniformly throughout the newly fertilized embryo, but becomes depleted in the posterior by the 4-cell stage. This MEX-3 patterning requires the CCCH zinc-finger protein MEX-5, the RNA Recognition Motif protein SPN-4, and the kinase PAR-4. Genetic and biochemical evidence suggests that MEX-5 binds to MEX-3 in the anterior of the embryo, protecting MEX-3 from degradation and allowing it to bind the pal-1 3'UTR and repress translation. MEX-3 that is not bound to MEX-5 becomes inactivated by par-4, then targeted for spn-4 dependent degradation. After the 4-cell stage, residual MEX-3 is degraded in somatic cells, and only persists in the germline precursors. To better understand regulation of mex-3, GFP was fused to MEX-3 or regions of MEX-3 and expressed in developing oocytes. GFP::MEX-3 expressed in this manner can replace endogenous MEX-3, but surprisingly is not asymmetrically localized at the 4-cell stage. These results indicate that GFP::MEX-3 retains asymmetric activity even in the absence of asymmetric protein localization. Neither the mex-3 3'UTR nor protein degradation at the 4-cell stage is strictly required. A region of MEX-3 containing a glutamine-rich region and potential ubiquitination and phosphorylation sites is sufficient for soma-germline asymmetry. Results from mex-5/6 and spn-4(RNAi) suggest two pathways for MEX-3 degradation, an early spn-4 dependent pathway and a later spn-4 independent pathway. These results indicate that mex-3 activity is regulated at multiple levels, leading to rapid and robust regulation in the quickly developing early embryo.

Assays for direct and indirect effects of C. elegans endo-siRNAs.

Ever since the discovery of the first microRNAs in C. elegans, increasing numbers of endogenous small RNAs have been discovered. Endogenous siRNAs (endo-siRNAs) have emerged in the last few years as a largely independent class of small RNAs that regulate endogenous gene expression, with mechanisms distinct from those of piRNAs and miRNAs. Quantification of these small RNAs and their effect on target RNAs is a powerful tool for the analysis of RNAi; however, detection of small RNAs can be difficult due to their small size and relatively low abundance. Here, we describe the novel FirePlex assay for directly detecting endo-siRNA levels in bulk, as well as an optimized qPCR method for detecting the effect of endo-siRNAs on gene targets. Intriguingly, the loss of endo-siRNAs frequently results in enhanced experimental RNAi. Thus, we also present an optimized method to assess the indirect impact of endo-siRNAs on experimental RNAi efficiency.

The DEAD box helicase RDE-12 promotes amplification of RNAi in cytoplasmic foci in C. elegans.

RNAi is a potent mechanism for downregulating gene expression. Conserved RNAi pathway components are found in animals, plants, fungi, and other eukaryotes. In C. elegans, the RNAi response is greatly amplified by the synthesis of abundant secondary small interfering RNAs (siRNAs). Exogenous double-stranded RNA is processed by Dicer and RDE-1/Argonaute into primary siRNA that guides target mRNA recognition. The RDE-10/RDE-11 complex and the RNA-dependent RNA polymerase RRF-1 then engage the target mRNA for secondary siRNA synthesis. However, the molecular link between primary siRNA production and secondary siRNA synthesis remains largely unknown. Furthermore, it is unclear whether the subcellular sites for target mRNA recognition and degradation coincide with sites where siRNA synthesis and amplification occur. In the C. elegans germline, cytoplasmic P granules at the nuclear pores and perinuclear Mutator foci contribute to target mRNA surveillance and siRNA amplification, respectively. We report that RDE-12, a conserved phenylalanine-glycine (FG) domain-containing DEAD box helicase, localizes in P granules and cytoplasmic foci that are enriched in RSD-6 but are excluded from the Mutator foci. Our results suggest that RDE-12 promotes secondary siRNA synthesis by orchestrating the recruitment of RDE-10 and RRF-1 to primary siRNA-targeted mRNA in distinct cytoplasmic compartments.

Natural RNA interference directs a heritable response to the environment.

RNA interference can induce heritable gene silencing, but it remains unexplored whether similar mechanisms play a general role in responses to cues that occur in the wild. We show that transient, mild heat stress in the nematode Caenorhabditis elegans results in changes in messenger RNA levels that last for more than one generation. The affected transcripts are enriched for genes targeted by germline siRNAs downstream of the piRNA pathway, and worms defective for germline RNAi are defective for these heritable effects. Our results demonstrate that a specific siRNA pathway transmits information about variable environmental conditions between generations.

The nuclear argonaute NRDE-3 contributes to transitive RNAi in Caenorhabditis elegans.

The Caenorhabditis elegans nuclear RNA interference defective (Nrde) mutants were identified by their inability to silence polycistronic transcripts in enhanced RNAi (Eri) mutant backgrounds. Here, we report additional nrde-3-dependent RNAi phenomena that extend the mechanisms, roles, and functions of nuclear RNAi. We show that nrde-3 mutants are broadly RNAi deficient and that overexpressing NRDE-3 enhances RNAi. Consistent with NRDE-3 being a dose-dependent limiting resource for effective RNAi, we find that NRDE-3 is required for eri-dependent enhanced RNAi phenotypes, although only for a subset of target genes. We then identify pgl-1 as an additional limiting RNAi resource important for eri-dependent silencing of a nonoverlapping subset of target genes, so that an nrde-3; pgl-1; eri-1 triple mutant fails to show enhanced RNAi for any tested gene. These results suggest that nrde-3 and pgl-1 define separate and independent limiting RNAi resource pathways. Limiting RNAi resources are proposed to primarily act via endogenous RNA silencing pathways. Consistent with this, we find that nrde-3 mutants misexpress genes regulated by endogenous siRNAs and incompletely silence repetitive transgene arrays. Finally, we find that nrde-3 contributes to transitive RNAi, whereby amplified silencing triggers act in trans to silence sequence-similar genes. Because nrde-dependent silencing is thought to act in cis to limit the production of primary transcripts, this result reveals an unexpected role for nuclear processes in RNAi silencing.

SID-5 is an endosome-associated protein required for efficient systemic RNAi in C. elegans.

In the nematode C. elegans, RNAi silencing signals are efficiently taken up from the environment and transported between cells and tissues. Previous studies implicating endosomal proteins in systemic RNAi lack conclusive evidence. Here, we report the identification and characterization of SID-5, a C. elegans endosome-associated protein that is required for efficient systemic RNAi in response to both ingested and expressed double-stranded RNA (dsRNA). SID-5 is detected in cytoplasmic foci that partially colocalize with GFP fusions of late endosomal proteins RAB-7 and LMP-1. Furthermore, knockdown of various endosomal proteins similarly relocalizes both SID-5 and LMP-1::GFP. Consistent with a non-cell-autonomous function, intestine-specific SID-5 expression restored body wall muscle (bwm) target gene silencing in response to ingested dsRNA. Finally, we show that sid-5 is required for the previously described sid-1-independent transport of ingested RNAi triggers across the intestine. Together, these data demonstrate that an endosome-associated protein, SID-5, promotes the transport of RNAi silencing signals between cells. Furthermore, SID-5 acts differently than the previously described SID-1, SID-2, and SID-3 proteins, thus expanding the systemic RNAi pathway.

Conserved tyrosine kinase promotes the import of silencing RNA into Caenorhabditis elegans cells.

RNA silencing in Caenorhabditis elegans is transmitted between cells by the transport of double-stranded RNA (dsRNA). The efficiency of such transmission, however, depends on both the cell type and the environment. Here, we identify systemic RNAi defective-3 (SID-3) as a conserved tyrosine kinase required for the efficient import of dsRNA. Without SID-3, cells perform RNA silencing well but import dsRNA poorly. Upon overexpression of SID-3, cells import dsRNA more efficiently than do wild-type cells and such efficient import of dsRNA requires an intact SID-3 kinase domain. The mammalian homolog of SID-3, activated cdc-42-associated kinase (ACK), acts in many signaling pathways that respond to environmental changes and is known to directly associate with endocytic vesicles, which have been implicated in dsRNA transport. Therefore, our results suggest that the SID-3/ACK tyrosine kinase acts as a regulator of RNA import into animal cells.

Uptake of extracellular double-stranded RNA by SID-2.

Ingested dsRNAs trigger RNA interference (RNAi) in many invertebrates, including the nematode Caenorhabditis elegans. Here we show that the C. elegans apical intestinal membrane protein SID-2 is required in C. elegans for the import of ingested dsRNA and that, when expressed in Drosophila S2 cells, SID-2 enables the uptake of dsRNAs. SID-2-dependent dsRNA transport requires an acidic extracellular environment and is selective for dsRNAs with at least 50 base pairs. Through structure-function analysis, we identify several SID-2 regions required for this activity, including three extracellular, positively charged histidines. Finally, we find that SID-2-dependent transport is inhibited by drugs that interfere with vesicle transport. Therefore, we propose that environmental dsRNAs are imported from the acidic intestinal lumen by SID-2 via endocytosis and are released from internalized vesicles in a secondary step mediated by the dsRNA channel SID-1. Similar multistep mechanisms may underlie the widespread observations of environmental RNAi.

A genomic bias for genotype-environment interactions in C. elegans.

The phenotype of an organism is determined by its genotype and environment. An interaction between these two arises from the differential effect of the environment on gene expression in distinct genotypes; however, the genomic properties identifying these are not well understood. Here we analyze the transcriptomes of five C. elegans strains (genotype) cultivated in five growth conditions (environment), and find that highly regulated genes, as distinguished by intergenic lengths, motif concentration, and expression levels, are particularly biased toward genotype-environment interactions. Sequencing these strains, we find that genes with expression variation across genotypes are enriched for promoter single-nucleotide polymorphisms (SNPs), as expected. However, genes with genotype-environment interactions do not significantly differ from background in terms of their promoter SNPs. Collectively, these results indicate that the highly regulated nature of particular genes predispose them for exhibiting genotype-environment interaction as a consequence of changes to upstream regulators. This observation may provide a deeper understanding into the origin of the extraordinary gene expression diversity present in even closely related species.

Vampiric isolation of extracellular fluid from Caenorhabditis elegans.

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ease of growth and small size. This small size, though, has disallowed a number of technical approaches found in other model systems. For example, blood transfusions in mammalian systems and grafting techniques in plants enable asking questions of circulatory system composition and signaling. The circulatory system of the worm, the pseudocoelom, has until recently been impossible to assay directly. To answer questions of intercellular signaling and circulatory system composition C. elegans researchers have traditionally turned to genetic analysis, cell/tissue specific rescue, and mosaic analysis. These techniques provide a means to infer what is happening between cells, but are not universally applicable in identification and characterization of extracellular molecules. Here we present a newly developed technique to directly assay the pseudocoelomic fluid of C. elegans. The technique begins with either genetic or physical manipulation to increase the volume of extracellular fluid. Afterward the animals are subjected to a vampiric reverse microinjection technique using a microinjection rig that allows fine balance pressure control. After isolation of extracellular fluid, the collected fluid can be assayed by transfer into other animals or by molecular means. To demonstrate the effectiveness of this technique we present a detailed approach to assay a specific example of extracellular signaling molecules, long dsRNA during a systemic RNAi response. Although characterization of systemic RNAi is a proof of principle example, we see this technique as being adaptable to answer a variety of questions of circulatory system composition and signaling.

The Influence of Competition Among C. elegans Small RNA Pathways on Development.

Small RNAs play a variety of regulatory roles, including highly conserved developmental functions. Caenorhabditis elegans not only possesses most known small RNA pathways, it is also an easy system to study their roles and interactions during development. It has been proposed that in C. elegans, some small RNA pathways compete for access to common limiting resources. The strongest evidence supporting this model is that disrupting the production or stability of endogenous short interfering RNAs (endo-siRNAs) enhances sensitivity to experimentally induced exogenous RNA interference (exo-RNAi). Here, we examine the relationship between the endo-siRNA and microRNA (miRNA) pathways, and find that, consistent with competition among these endogenous small RNA pathways, endo-siRNA pathway mutants may enhance miRNA efficacy. Furthermore, we show that exo-RNAi may also compete with both endo-siRNAs and miRNAs. Our data thus provide support that all known Dicer-dependent small RNA pathways may compete for limiting common resources. Finally, we observed that both endo-siRNA mutants and animals experiencing exo-RNAi have increased expression of miRNA-regulated stage-specific developmental genes. These observations suggest that perturbing the small RNA flux and/or the induction of exo-RNAi, even in wild-type animals, may impact development via effects on the endo-RNAi and microRNA pathways.