“Caenorhabditis elegans SID-2 is required for intestinal uptake of ingested double-stranded RNA.”

The uptake of ingested dsRNAs into intestinal cells has been shown to require the SID-2 transmembrane protein in C. elegans. … In a screen for C. elegans mutants that are specifically defective in the uptake of externally administered dsRNAs, but not in the downstream response (Sid phenotype, for systemic-interference-defective), Winston et al. [4] found mutants at two loci, named sid-1 and sid-2. The sid-1 gene encodes a transmembrane protein that is necessary for spread of the RNAi signal within the organism [4], whereas the sid-2 gene is required for the primary uptake of dsRNAs into intestinal cells [5].Background The nematode Caenorhabditis elegans is able to take up external double-stranded RNAs (dsRNAs) and mount an RNA interference response, leading to the inactivation of specific gene expression. The uptake of ingested dsRNAs into intestinal cells has been shown to require the SID-2 transmembrane protein in C. elegans. By contrast, C. briggsae was shown to be naturally insensitive to ingested dsRNAs, yet could be rendered sensitive by transgenesis with the C. elegans sid-2 gene. Here we aimed to elucidate the evolution of the susceptibility to external RNAi in the Caenorhabditis genus. Principal Findings … RNA interference is the inactivation of gene expression induced by double-stranded RNAs (dsRNAs) [1]. The molecular mechanism by which dsRNAs introduced into a cell induce degradation of the corresponding mRNA has been well documented in the nematode Caenorhabditis elegans. In this species, dsRNAs do not need to be injected, but are able to enter cells when the animal is either soaked in a dsRNA preparation [2], or is fed E. coli bacteria that express dsRNAs from a plasmid [3]. In a screen for C. elegans mutants that are specifically defective in the uptake of externally administered dsRNAs, but not in the downstream response (Sid phenotype, for systemic-interference-defective), Winston et al. [4] found mutants at two loci, named sid-1 and sid-2. The sid-1 gene encodes a transmembrane protein that is necessary for spread of the RNAi signal within the organism [4], whereas the sid-2 gene is required for the primary uptake of dsRNAs into intestinal cells [5]. Interestingly, C. briggsae, a close relative of C. elegans, encodes a divergent form of sid-2, which appears unable to take up dsRNAs. The inability of C. briggsae to take up ingested dsRNAs can be rescued by expression of the C. elegans sid-2 (Cel-sid-2) gene in C. briggsae [5]. Winston et al. …

Here we report the identification and characterization of SID-2, an intestinal luminal transmembrane protein required for environmental RNAi in C. elegans. … We find that SID-2 is a transmembrane protein expressed in the intestine and localized to the apical (luminal) membrane, indicating that the gut is the only significant route of entry for dsRNA into the animal. … Thus, sid-2 is required for the uptake of environmental dsRNA but, unlike sid-1, is not required for the subsequent spread of dsRNA between cells.… elegans as well. Furthermore, in C. elegans, dsRNA present in the environment or expressed in ingested bacteria is sufficient to trigger RNAi (environmental RNAi). Ingestion and soaking delivery of dsRNA has also been described for other invertebrates. Here we report the identification and characterization of SID-2, an intestinal luminal transmembrane protein required for environmental RNAi in C. elegans. SID-2, when expressed in the environmental RNAi defective species Caenorhabditis briggsae, confers environmental RNAi. Translocation of nucleic acids across cellular membranes is associated with viral infection, bacterial conjugation, and transport of nuclear encoded tRNAs into mitochondria (1–3). … This observation suggested that environmental dsRNA might enter the animal via these sid-1 expressing cells. Here we describe sid-2, a gene specifically required for uptake of silencing information (hereafter assumed to be dsRNA) from the environment. We find that SID-2 is a transmembrane protein expressed in the intestine and localized to the apical (luminal) membrane, indicating that the gut is the only significant route of entry for dsRNA into the animal. Remarkably, SID-2 function is not conserved in the related nematode Caenorhabditis briggsae, but expression of C. elegans SID-2 is sufficient to confer environmental RNAi on C. briggsae. Based on our analysis of the roles of sid-1 and -2, we propose that environmental RNAi in C. … However, similar to wild-type and in contrast to sid-1 mutants, sid-2(qt13) worms were fully sensitive to systemic RNAi initiated by injection or transgenic expression of dsRNA targeting somatic and germ-line-expressed genes (Tables 1 and 2; Fig. 1). Thus, sid-2 is required for the uptake of environmental dsRNA but, unlike sid-1, is not required for the subsequent spread of dsRNA between cells. Characterization of sid-2 deficiency on RNAi of a germ-line-expressed gene

Here we report the identification and characterization of SID-2, an intestinal luminal transmembrane protein required for environmental RNAi in C. elegans. … We find that SID-2 is a transmembrane protein expressed in the intestine and localized to the apical (luminal) membrane, indicating that the gut is the only significant route of entry for dsRNA into the animal. … Thus, sid-2 is required for the uptake of environmental dsRNA but, unlike sid-1, is not required for the subsequent spread of dsRNA between cells.… elegans as well. Furthermore, in C. elegans, dsRNA present in the environment or expressed in ingested bacteria is sufficient to trigger RNAi (environmental RNAi). Ingestion and soaking delivery of dsRNA has also been described for other invertebrates. Here we report the identification and characterization of SID-2, an intestinal luminal transmembrane protein required for environmental RNAi in C. elegans. SID-2, when expressed in the environmental RNAi defective species Caenorhabditis briggsae, confers environmental RNAi. Translocation of nucleic acids across cellular membranes is associated with viral infection, bacterial conjugation, and transport of nuclear encoded tRNAs into mitochondria (1–3). … This observation suggested that environmental dsRNA might enter the animal via these sid-1 expressing cells. Here we describe sid-2, a gene specifically required for uptake of silencing information (hereafter assumed to be dsRNA) from the environment. We find that SID-2 is a transmembrane protein expressed in the intestine and localized to the apical (luminal) membrane, indicating that the gut is the only significant route of entry for dsRNA into the animal. Remarkably, SID-2 function is not conserved in the related nematode Caenorhabditis briggsae, but expression of C. elegans SID-2 is sufficient to confer environmental RNAi on C. briggsae. Based on our analysis of the roles of sid-1 and -2, we propose that environmental RNAi in C. … However, similar to wild-type and in contrast to sid-1 mutants, sid-2(qt13) worms were fully sensitive to systemic RNAi initiated by injection or transgenic expression of dsRNA targeting somatic and germ-line-expressed genes (Tables 1 and 2; Fig. 1). Thus, sid-2 is required for the uptake of environmental dsRNA but, unlike sid-1, is not required for the subsequent spread of dsRNA between cells. Characterization of sid-2 deficiency on RNAi of a germ-line-expressed gene

SID-2 is a transmembrane protein localized to the apical membrane of intestinal cells and required for dsRNA uptake from the intestinal lumen.… elegans, and then imported into the cytosol of other cells where processing of dsRNA occurs to generate small interfering RNA. 9 Forward genetic screens identified genes, each of which is involved in a respective step of feeding RNAi. 2, 10 The dsRNA importer systemic RNAi defective-1 (SID-1) is required for the import of dsRNA into the cytosol but dispensable for the export of dsRNA. 11, 12 SID-2 is a transmembrane protein localized to the apical membrane of intestinal cells and required for dsRNA uptake from the intestinal lumen. 13, 14 SID-3 and NCK-1/SID-4, homologs of ACK1 kinase and its interactor NCK protein, respectively, 15, 16 and an ENTH domain protein RSD-3 10, 17 have been shown to be involved in the import of silencing signals from the pseudocoelom. Regulators of the endocytic pathway that mediate dsRNA uptake into Drosophila S2 cells are also suggested to be involved in cell entry of dsRNA in C. elegans. 18 SID-5 is a single-pass transmembrane protein and associates with late endosomes. …

whereas the sid-2 gene is required for the primary uptake of dsRNAs into intestinal cells [5]. … As in [5], we could ‘‘rescue’’ C. briggsae insensitivity to ingested dsRNAs with Cel-sid-2, using a simple PCR product of the Cel-sid-2 gene instead of a fusion construct to GFP (see Materials and Methods). … The response of these two species to ingested dsRNAs can be rescued by transgenesis with the Cel-sid-2 gene.… [4] found mutants at two loci, named sid 1 and sid-2. The sid-1 gene encodes a transmembrane protein that is necessary for spread of the RNAi signal within the organism [4], whereas the sid-2 gene is required for the primary uptake of dsRNAs into intestinal cells [5]. Interestingly, C. briggsae, a close relative of C. elegans, encodes a divergent form of sid-2, which appears unable to take up dsRNAs. The inability of C. briggsae to take up ingested dsRNAs can be rescued by expression of the C. elegans sid-2 (Cel-sid-2) gene in C. briggsae [5]. Winston et al. … Results for C. elegans are shown as positive control for the efficiency of the dsRNA treatment. doi:10.1371/journal.pone.0029811.g004 PLoS ONE | www.plosone.org 8 January 2012 | Volume 7 | Issue 1 | e29811 we thus wished to obtain a non-Roller transgenic line that would help in the scoring of many phenotypes. As in [5], we could ‘‘rescue’’ C. briggsae insensitivity to ingested dsRNAs with Cel-sid-2, using a simple PCR product of the Cel-sid-2 gene instead of a fusion construct to GFP (see Materials and Methods). We first obtained an extra-chromosomal transgenic line in the C. briggsae AF16 wild background expressing Cel-sid-2 and the Cel-myo-2::DsRed2 marker. After irradiation and backcrosses to C. briggsae AF16, we derived two integrated transgenic strains, JU1018 and JU1076. … We will review the situation in the relevant evolutionary groups successively (Figure 3). In the clade including C. briggsae and C. remanei, the loss of the ability to respond to ingested Oti-actin dsRNAs corresponds to the apparent inability to respond to any external dsRNAs, yet with the ability to respond to internal dsRNAs. The response of these two species to ingested dsRNAs can be rescued by transgenesis with the Cel-sid-2 gene. In the sister clade, C. brenneri was previously shown to have further lost the capacity to mount a systemic response to internal dsRNAs, when they were injected into the body cavity instead of the gonad [13]. The inability of C. sp. …

Here we show that the C. elegans apical intestinal membrane protein SID-2 is required in C. elegans for the import of ingested dsRNA … Using confocal microscopy, we observed cy5-labeled structures within the intestinal cells of control animals but not in any of the sid-2(gk505) mutants (Figure 1). Therefore, SID-2 is required either for the initial uptake of dsRNA and/or its accumulation in distinct intestinal compartments. … SID-2 is required to internalize environmental dsRNAs into C. elegans. 500 base pair (bp) cy5-labeled dsRNA fed to C. elegans could only be visualized intracellularly in sid-2(+) animals (observed in 6/15 sid-2(+) animals, 0/15 sid-2(−) animals).Summary 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, 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. … elegans sid-2 mutants can import any extracellular dsRNA. We soaked sid-2(gk505) deletion mutants and positive-control animals expressing a rescuing translational fusion of SID-2 and GFP (SID-2::GFP) (Winston et al., 2007) in cy5-labeled 500 base pair (bp) dsRNA for one to two hours. Using confocal microscopy, we observed cy5-labeled structures within the intestinal cells of control animals but not in any of the sid-2(gk505) mutants (Figure 1). Therefore, SID-2 is required either for the initial uptake of dsRNA and/or its accumulation in distinct intestinal compartments. We did not observe any significant intracellular overlap of the cy5 signal and SID-2::GFP. Figure 1. Open in a new tab SID-2 is required to internalize environmental dsRNAs into C. elegans. 500 base pair (bp) cy5-labeled dsRNA fed to C. elegans could only be visualized intracellularly in sid-2(+) animals (observed in 6/15 sid-2(+) animals, 0/15 sid-2(−) animals). SID-2 enables pH-dependent dsRNA uptake when expressed in Drosophila S2 cells

SID-2 is an intestinally expressed transmembrane protein, present at the intestinal lumen, that selectively endocytoses ingested dsRNA (Winston et al. 2007; McEwan et al. 2012). While SID-2 is required only for feeding RNAi, SID-1 is also required for feeding RNAi; thus, it is presumed that SID-1 releases endocytosed dsRNA into the cytoplasm to initiate RNAi.… SID-1 is a dsRNA channel protein that selectively transports long dsRNA into cells (Feinberg and Hunter 2003; Shih and Hunter 2011). In sid-1 mutants, systemic RNAi is undetectable, but expressed or injected dsRNA can cause robust autonomous RNAi (Winston et al. 2002). Genetic mosaic and tissue-specific rescue experiments demonstrate that SID-1 is required for import but not export of silencing information (presumably dsRNA) (Winston et al. 2002; Jose et al. 2009; Whangbo et al. 2017). SID-2 is an intestinally expressed transmembrane protein, present at the intestinal lumen, that selectively endocytoses ingested dsRNA (Winston et al. 2007; McEwan et al. 2012). While SID-2 is required only for feeding RNAi, SID-1 is also required for feeding RNAi; thus, it is presumed that SID-1 releases endocytosed dsRNA into the cytoplasm to initiate RNAi. SID-3 is a broadly expressed ACK1 tyrosine kinase homolog (Jose et al. 2012). sid-3 mutants are partially defective for the import of silencing signals. The requirement of a signal transduction protein suggests that environmental or physiological conditions may regulate systemic RNAi. SID-5 is a small novel protein associated with late endosomes (Hinas et al. 2012). …

Here we report the identification and characterization of SID-2, an intestinal luminal transmembrane protein required for environmental RNAi in C. elegans. … We find that SID-2 is a transmembrane protein expressed in the intestine and localized to the apical (luminal) membrane, indicating that the gut is the only significant route of entry for dsRNA into the animal. … These results suggest that sid-2 enables import of ingested dsRNA from the intestinal lumen.… elegans as well. Furthermore, in C. elegans, dsRNA present in the environment or expressed in ingested bacteria is sufficient to trigger RNAi (environmental RNAi). Ingestion and soaking delivery of dsRNA has also been described for other invertebrates. Here we report the identification and characterization of SID-2, an intestinal luminal transmembrane protein required for environmental RNAi in C. elegans. SID-2, when expressed in the environmental RNAi defective species Caenorhabditis briggsae, confers environmental RNAi. Keywords: dsRNA, transmembrane, intestine lumen … This observation suggested that environmental dsRNA might enter the animal via these sid-1 expressing cells. Here we describe sid-2, a gene specifically required for uptake of silencing information (hereafter assumed to be dsRNA) from the environment. We find that SID-2 is a transmembrane protein expressed in the intestine and localized to the apical (luminal) membrane, indicating that the gut is the only significant route of entry for dsRNA into the animal. Remarkably, SID-2 function is not conserved in the related nematode Caenorhabditis briggsae, but expression of C. elegans SID-2 is sufficient to confer environmental RNAi on C. briggsae. Based on our analysis of the roles of sid-1 and -2, we propose that environmental RNAi in C. … Consistent with the restricted expression, mosaic analysis of sid-2 confirmed that sid-2 activity is not required in muscle cells for uptake of silencing information into muscle cells (SI Fig. 6). These results suggest that sid-2 enables import of ingested dsRNA from the intestinal lumen. SID-1 and SID-2 Are Both Required for the Import of Environmental dsRNA into Intestinal Cells.

Here we show that the C. elegans apical intestinal membrane protein SID-2 is required in C. elegans for the import of ingested dsRNA … We soaked sid-2(gk505) deletion mutants and positive-control animals expressing a rescuing translational fusion of SID-2 and GFP (SID-2::GFP) (Winston et al., 2007) in cy5-labeled 500 base pair (bp) dsRNA for one to two hours. Using confocal microscopy, we observed cy5-labeled structures within the intestinal cells of control animals but not in any of the sid-2(gk505) mutants (Figure 1). Therefore, SID-2 is required either for the initial uptake of dsRNA and/or its accumulation in distinct intestinal compartments.Summary 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, 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. … ### SID-2 is required for environmental dsRNA to accumulate in C. elegans intestinal cells To determine whether SID-2 functions during the initial uptake of dsRNA from the intestinal lumen or for later processing/transport of already internalized dsRNA, we first tested whether C. elegans sid-2 mutants can import any extracellular dsRNA. We soaked sid-2(gk505) deletion mutants and positive-control animals expressing a rescuing translational fusion of SID-2 and GFP (SID-2::GFP) (Winston et al., 2007) in cy5-labeled 500 base pair (bp) dsRNA for one to two hours. Using confocal microscopy, we observed cy5-labeled structures within the intestinal cells of control animals but not in any of the sid-2(gk505) mutants (Figure 1). Therefore, SID-2 is required either for the initial uptake of dsRNA and/or its accumulation in distinct intestinal compartments. We did not observe any significant intracellular overlap of the cy5 signal and SID-2::GFP. Open in a separate window

The uptake of ingested dsRNAs into intestinal cells has been shown to require the SID-2 transmembrane protein in C. elegans. … the sid-2 gene is required for the primary uptake of dsRNAs into intestinal cellsAbstract The nematode Caenorhabditis elegans is able to take up external double-stranded RNAs (dsRNAs) and mount an RNA interference response, leading to the inactivation of specific gene expression. The uptake of ingested dsRNAs into intestinal cells has been shown to require the SID-2 transmembrane protein in C. elegans. By contrast, C. briggsae was shown to be naturally insensitive to ingested dsRNAs, yet could be rendered sensitive by transgenesis with the C. elegans sid-2 gene. Here we aimed to elucidate the evolution of the susceptibility to external RNAi in the Caenorhabditis genus. Principal Findings: We study the sensitivity of many new species of Caenorhabditis to ingested dsRNAs matching a conserved actin gene sequence from the nematode Oscheius tipulae. … coli bacteria that express dsRNAs from a plasmid [3] . In a screen for C. elegans mutants that are specifically defective in the uptake of externally administered dsRNAs, but not in the downstream response (Sid phenotype, for systemic-interferencedefective), Winston et al. [4] found mutants at two loci, named sid-1 and sid-2. The sid-1 gene encodes a transmembrane protein that is necessary for spread of the RNAi signal within the organism [4] , whereas the sid-2 gene is required for the primary uptake of dsRNAs into intestinal cells [5] . Interestingly, C. briggsae, a close relative of C. elegans, encodes a divergent form of sid-2, which appears unable to take up dsRNAs. The inability of C. briggsae to take up ingested dsRNAs can be rescued by expression of the C. elegans sid-2 (Cel-sid-2) gene in C. briggsae [5] . Winston et al. …

The uptake of ingested dsRNAs into intestinal cells has been shown to require the SID-2 transmembrane protein in C. elegans. … the sid-2 gene is required for the primary uptake of dsRNAs into intestinal cellsAbstract The nematode Caenorhabditis elegans is able to take up external double-stranded RNAs (dsRNAs) and mount an RNA interference response, leading to the inactivation of specific gene expression. The uptake of ingested dsRNAs into intestinal cells has been shown to require the SID-2 transmembrane protein in C. elegans. By contrast, C. briggsae was shown to be naturally insensitive to ingested dsRNAs, yet could be rendered sensitive by transgenesis with the C. elegans sid-2 gene. Here we aimed to elucidate the evolution of the susceptibility to external RNAi in the Caenorhabditis genus. Principal Findings: We study the sensitivity of many new species of Caenorhabditis to ingested dsRNAs matching a conserved actin gene sequence from the nematode Oscheius tipulae. … coli bacteria that express dsRNAs from a plasmid [3] . In a screen for C. elegans mutants that are specifically defective in the uptake of externally administered dsRNAs, but not in the downstream response (Sid phenotype, for systemic-interferencedefective), Winston et al. [4] found mutants at two loci, named sid-1 and sid-2. The sid-1 gene encodes a transmembrane protein that is necessary for spread of the RNAi signal within the organism [4] , whereas the sid-2 gene is required for the primary uptake of dsRNAs into intestinal cells [5] . Interestingly, C. briggsae, a close relative of C. elegans, encodes a divergent form of sid-2, which appears unable to take up dsRNAs. The inability of C. briggsae to take up ingested dsRNAs can be rescued by expression of the C. elegans sid-2 (Cel-sid-2) gene in C. briggsae [5] . Winston et al. …

SID-2 is an intestinal lumenal membrane protein that is required for the initial uptake of dsRNA from the environment, but not for intercellular transport of silencing signals [7, 12].sid-5 is required for systemic RNAi in C. elegans and is predicted to encode a transmembrane protein SID-1 and SID-2 are two previously identified dsRNA transport proteins that are required for the uptake and spreading of RNAi silencing signals in C. elegans [3, 7]. SID-1 is a broadly expressed dsRNA channel that is required for import of silencing signals into all cells [3, 6, 9–11]. SID-2 is an intestinal lumenal membrane protein that is required for the initial uptake of dsRNA from the environment, but not for intercellular transport of silencing signals [7, 12]. In addition, the recently identified SID-3 is an ACK1-like non-receptor tyrosine kinase required for the efficient import of dsRNA [8]. The same screen Correspondence: andrea.hinas@imbim.uu.se, +46 18 471 4476 (A.H.), hunter@mcb.harvard.edu, +1(617) 495 8309 (C.P.H.).

Here we show that the C. elegans apical intestinal membrane protein SID-2 is required in C. elegans for the import of ingested dsRNA … SID-2 is required to internalize environmental dsRNAs into C. elegans. 500 base pair (bp) cy5-labeled dsRNA fed to C. elegans could only be visualized intracellularly in sid-2(+) animals (observed in 6/15 sid-2(+) animals, 0/15 sid-2(−) animals).Abstract 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. … Figure 1 SID-2 is required to internalize environmental dsRNAs into C. elegans. 500 base pair (bp) cy5-labeled dsRNA fed to C. elegans could only be visualized intracellularly in sid-2(+) animals (observed in 6/15 sid-2(+) animals, 0/15 sid-2(−) animals). Figure 2

Thus rsd-4 and rsd-8 mutants appear to have a complete defect in the cellular uptake of dsRNA. In contrast, rsd-2, rsd-3, and rsd-6 mutants appear not defective in the initial uptake of dsRNA from the gut into somatic tissues but are unable to further distribute this dsRNA to the germline. … The rsd genes can be divided into two classes based on phenotype. Class I mutants are not susceptible to RNAi via feeding of either germline or somatic genes, whereas Class II mutants are sensitive to RNAi of somatic genes, but not of germline genes.… The second class, composed of rsd-2, rsd-3, and rsd-6, also showed no phenotype when fed on dsRNA against a germline gene such as pos-1. However, RNAi directed against somatic genes was still able to produce somatic phenotypes in these mutants. This effect was reproducible for a number of different somatic and germline genes (Table 1). In addition, this effect was also seen when transgenic animals expressing GFP in all tissues were fed on RNAi food targeting GFP (Figures 1B and 1C). Thus rsd-4 and rsd-8 mutants appear to have a complete defect in the cellular uptake of dsRNA. In contrast, rsd-2, rsd-3, and rsd-6 mutants appear not defective in the initial uptake of dsRNA from the gut into somatic tissues but are unable to further distribute this dsRNA to the germline. Figure 1 Identification and Characterization of RNAi Spreading-Defective Mutants … The rsd Mutants Fall into Two Classes Based on Phenotype The rsd genes can be divided into two classes based on phenotype. Class I mutants are not susceptible to RNAi via feeding of either germline or somatic genes, whereas Class II mutants are sensitive to RNAi of somatic genes, but not of germline genes. Phenotypes based on that used by Wormbase (www.wormbase.org). Abbreviations: twitch, uncontrollable body muscle twitching; unc, uncoordinated motion; emb, embryonic lethality; ste, sterility. - Reprints - Open table in a new tab

In the nematode Caenorhabditis elegans, a transmembrane protein named systemic RNAi deficient-2 (sid-2) is required for dsRNA uptake from the environment through the intestine. … The single-pass trans-membrane protein SID-2 is required for the uptake of dsRNA from the environment into the gut (McEwan et al., 2012; Winston et al., 2007). Genetic mutations in sid-2 cause resistance to RNAi by feeding (Winston et al., 2007).Abstract dsRNA uptake from the environment is a common phenomenon in many invertebrates and can be harnessed for acquiring pest control. In the nematode Caenorhabditis elegans, a transmembrane protein named systemic RNAi deficient-2 (sid-2) is required for dsRNA uptake from the environment through the intestine. Furthermore, the tyrosine-protein kinase sid-3 promotes import dsRNA. However, the molecular mechanism underlying environmental RNA uptake and its natural phenotypic roles are largely unknown. Here I characterized the intracellular properties of SID-2 and its role during the nematode development. I determined SID-2 localization at the intestinal apical membrane and the trans-Golgi-network (TGN). … dsRNA uptake at the apical intestinal membrane The single-pass trans-membrane protein SID-2 is required for the uptake of dsRNA from the environment into the gut (McEwan et al., 2012; Winston et al., 2007). Genetic mutations in sid-2 cause resistance to RNAi by feeding (Winston et al., 2007). However, in these animals, the downstream RNAi Transgene A B C D E F G Pharynx GFP hpGFP GFP hpGFP GFP hpGFP GFP hpGFP GFP hpGFP GFP Bodywall GFP GFP GFP GFP GFP GFP GFP Genotype wt wt wt wt wt rde-1 (-/-) sid-1 (-/-)

The transmembrane protein SID-1 is essential for systemic RNAi in C. elegans because it allows the passive cellular uptake of dsRNA … uptake of dsRNA from the intestinal lumen, which requires the sid-2 gene (ref. 20), might be thought sufficient for intestinal RNAi. Winston et al.20, however, also found that feeding RNAi for intestinally expressed genes did not occur without sid-1.Since its discovery1, RNA interference (RNAi) has served as a powerful tool to study gene function, especially in Caenorhabditis elegans. C. elegans is unusual in that it exhibits systemic RNAi; double stranded (ds) RNA in the environment can enter and spread throughout the worm to silence the targeted gene2,3. RNAi occurs when worms are soaked in solutions of dsRNA or fed bacteria expressing dsRNA (feeding RNAi), yielding a powerful tool for reverse genetics in this organism4,5. The transmembrane protein SID-1 is essential for systemic RNAi in C. elegans because it allows the passive cellular uptake of dsRNA6. Drosophila possesses robust cell-autonomous RNAi but lacks both systemic RNAi and a SID-1 homolog7. Drosophila cells6,7 or mouse embryonic stem cells8 expressing the C. elegans SID-1, however, respond to dsRNA in the media. Feeding RNAi is robust in virtually all cells in C. elegans except neurons5,9. In contrast, RNAi occurs in neurons when dsRNA is produced within the neurons themselves10. … We used wild type (N2) as controls that do not express Punc-119sid-1(+) and strain TU3270 as controls that do. sid-1 mutants were included as negative controls. Values represent the mean ± S.E.M. of nine plates, 50 worms scored per plate. The lack of a response in the intestine is curious since uptake of dsRNA from the intestinal lumen, which requires the sid-2 gene (ref. 20), might be thought sufficient for intestinal RNAi. Winston et al.20, however, also found that feeding RNAi for intestinally expressed genes did not occur without sid-1. These results could indicate either that SID-1 is needed for the initial transport through the intestine or that SID-1 activity is needed for RNAi in the intestine, perhaps by mediating transport of dsRNA from the pseudocoelom. The finding that RNAi is observed when sid-1 mutants express sid-1(+) in muscle21 or in the TRNs (see below), suggests that SID-1 is not needed for the initial transport of dsRNA from the intestinal lumen. …

Unlike plants, C. elegans and Planaria can uptake dsRNA from the environment by feeding or soaking in a dsRNA solution. … a transmembrane protein called SID-1 has been identified in a forward genetic screen that is required for systemic RNA interference (RNAi) in C. elegansSystemic RNAi in animals Although first identified in plants, systemic RNA silencing also occurs in diverse animals including worms (C. elegans; Fire et al, 1998), insects (Tribolium; Bucher et al, 2002) and Planaria (Newmark et al, 2003). Unlike plants, C. elegans and Planaria can uptake dsRNA from the environment by feeding or soaking in a dsRNA solution. The exogenously supplied or microinjected dsRNA can trigger sequence-specific gene silencing in the treated and adjacent tissues and silence the progeny of injected animals. Thus, the long dsRNA or its derivatives must move across the cell membrane from cell-to-cell, unlike the situation in plants where the movement of the silencing signal occurs through a shared cytoplasm. This phenomenon implies the need for membrane-bound transporters and, indeed, a transmembrane protein called SID-1 has been identified in a forward genetic screen that is required for systemic RNA interference (RNAi) in C. elegans (Winston et al, 2002). SID-1 homologues exist is most animals but not plants, suggesting that systemic RNAi may also occur in other animals including mammals (Winston et al, 2002; Wolfrum et al, 2007). Since SID-1 has higher affinity for longer dsRNA than sRNA duplexes (Feinberg and Hunter, 2003), it is highly likely that long dsRNA is the main component of the RNAi-associated silencing signal in worms. …

Here we characterise the environmental dsRNA receptor SID-2 to understand the biological function of dsRNA uptake in Caenorhabditis elegans. First we determine that SID-2 localises to the apical membrane and the trans-Golgi-network (TGN) in the intestine, implicating the TGN as a central cellular compartment for environmental dsRNA uptake. … One such mutant defined an intestinal dsRNA receptor called Systemic RNA Interference Defective-2 (SID-2) is essential for the uptake of artificial dsRNA [26] . … Finally, all strains with a sid-2 mutation had a hatching rate of 100% indicating that they were unable to take up dsRNA (S2C Fig).Therefore, we concluded that pyr-1 mutants can take up dsRNA, and sid-2 does not enhance dsRNA uptake for nutritional purposes.… Many animal species can take-up double-stranded RNA (dsRNA) from the environment. Environmental dsRNA changes gene expression through RNA interference (RNAi). While environmental RNAi is used as a laboratory tool, e.g. in nematodes, planaria and insects, its biological role remains enigmatic. Here we characterise the environmental dsRNA receptor SID-2 to understand the biological function of dsRNA uptake in Caenorhabditis elegans. First we determine that SID-2 localises to the apical membrane and the trans-Golgi-network (TGN) in the intestine, implicating the TGN as a central cellular compartment for environmental dsRNA uptake. We demonstrate that SID-2 is irrelevant for nucleotide uptake from the environment as a nutritional (nitrogen) source. Instead RNA profiling and high-resolution live imaging revealed a new biological function for sid-2 in growth and phenotypic plasticity. … Mutations blocking environmental RNAi have been identified in forward genetic screens in the model organism C. elegans and have led to a mechanistic understanding of artificial dsRNA uptake, transport and processing [25] . One such mutant defined an intestinal dsRNA receptor called Systemic RNA Interference Defective-2 (SID-2) is essential for the uptake of artificial dsRNA [26] . Biochemical studies showed it is able to mediate the uptake of dsRNA and single stranded RNA forming only partial dsRNA structures in form of hairpins, but not the chemically similar DNA [27] . … Next, we fed RNAi against POsterior Segregation-1 (pos-1 ), a gene essential for embryo development, again in the presence of exogenous Uracil. The drastically reduced hatching (≈ 10%) of wild-type and pyr-1 mutant animals hatched, showing that animals are capable to take up dsRNA and mount an RNAi response. Finally, all strains with a sid-2 mutation had a hatching rate of 100% indicating that they were unable to take up dsRNA (S2C Fig).Therefore, we concluded that pyr-1 mutants can take up dsRNA, and sid-2 does not enhance dsRNA uptake for nutritional purposes.sid-2 reduces growth rate but increases phenotypic plasticityBecause artificial environmental dsRNA can induce gene regulation, naturally occurring dsRNA might induce directly or indirectly induce gene expression changes, which we can measure. …

According to the structures and functions of SID-1 and SID-2, SID-1 may act as dsRNA channel or a component of channel for uptaking dsRNAs, and SID-2 may act as a dsRNA receptor or a component of receptor to aid the intestinal uptaking by SID-1 of environmental dsRNAs in intestinal lumen. Indeed, it is true that sid-2 is required only for the uptaking of exogenous dsRNAs but is not necessary for the spreading of RNAi signal from intestinal cells to other cells.… elegans, C. briggsae and C. remanei [8] . sid-1 is expressed in most non-neuronal cells including intestinal cells. sid-2 is expressed in intestinal cells, and SID-2::GFP fusion localizes to intestinal cell apical membranes. Heterologous expression of either SID-1 or SID-2 in Drosophila melanogaster S2 cells led to cells gaining the ability of importing dsRNA from culture medium [12, 13] . According to the structures and functions of SID-1 and SID-2, SID-1 may act as dsRNA channel or a component of channel for uptaking dsRNAs, and SID-2 may act as a dsRNA receptor or a component of receptor to aid the intestinal uptaking by SID-1 of environmental dsRNAs in intestinal lumen. Indeed, it is true that sid-2 is required only for the uptaking of exogenous dsRNAs but is not necessary for the spreading of RNAi signal from intestinal cells to other cells. Conversely, sid-1 is required for both the initial import of dsRNA into worms and the spread of systemic RNAi signal, but not necessary for cells to export systemic RNAi signal [14] . SID-2-dependent dsRNA uptaking requires an acidic extracellular environment in intestinal lumen, and is selective for dsRNAs 50 base pairs or longer. SID-2-dependent transport could be suppressed inhibitory drugs of vesicle transportation. …

Here we report the identification and characterization of SID-2, an intestinal luminal transmembrane protein required for environmental RNAi in C. elegans.… In plants, this signal is known to be a form of dsRNA, and the signal is most likely a form of dsRNA in C. elegans as well. Furthermore, in C. elegans, dsRNA present in the environment or expressed in ingested bacteria is sufficient to trigger RNAi (environmental RNAi). Ingestion and soaking delivery of dsRNA has also been described for other invertebrates. Here we report the identification and characterization of SID-2, an intestinal luminal transmembrane protein required for environmental RNAi in C. elegans. SID-2, when expressed in the environmental RNAi defective species Caenorhabditis briggsae, confers environmental RNAi. - GENBANK/AY466439

The second largest complementation group recovered in the original screen identified SID-2, a single-pass transmembrane protein strongly expressed in the gut (Winston et al. 2002 and in prep.) . This restricted expression pattern corresponds to the phenotype observed in sid-2 mutants, which are only defective for environmental RNAi, i.e., RNAi triggered by soaking in dsRNA or ingesting bacteria expressing dsRNA. Consequently, sid-2 mutants are fully sensitive to systemic RNAi triggered by transgene expression or microinjection into any tissues.SID-2 The second largest complementation group recovered in the original screen identified SID-2, a single-pass transmembrane protein strongly expressed in the gut (Winston et al. 2002 and in prep.) . This restricted expression pattern corresponds to the phenotype observed in sid-2 mutants, which are only defective for environmental RNAi, i.e., RNAi triggered by soaking in dsRNA or ingesting bacteria expressing dsRNA. Consequently, sid-2 mutants are fully sensitive to systemic RNAi triggered by transgene expression or microinjection into any tissues. Topology-determining experiments show that the predicted TM domain separates an extracellular aminoterminal domain from an intracellular carboxy-terminal domain. Fusing GFP to the intracellular carboxyl terminus of SID-2 (SID-2-C::GFP) produces a fusion gene that fully rescues the sid-2 environmental RNAi defect and strongly localizes GFP to intracellular and surfacelocalized membrane structures (Fig. 5 ). To determine whether environmental RNAi sensitivity arose in C. …

William M. Winston, Marie E. Sutherlin, Amanda J. Wright, Evan H. Feinberg, Craig P. Hunter. Caenorhabditis elegans SID-2 is required for environmental RNA interference. Proceedings of the National Academy of Sciences. 2007;104(25):10565-10570. doi:10.1073/pnas.0611282104.… - Christian E. Rocheleau, Kevin Cullison, Kai Huang, Yelena Bernstein, Annina C. Spilker, Meera V. Sundaram. The Caenorhabditis elegans ekl (Enhancer of ksr-1 Lethality) Genes Include Putative Components of a Germline Small RNA Pathway. Genetics. 2008;178(3):1431-1443. doi:10.1534/genetics.107.084608. - Graham R. Martin. Eye structure and amphibious foraging in albatrosses. Proceedings of the Royal Society B Biological Sciences. 1998;265(1397):665-671. doi:10.1098/rspb.1998.0345. - William M. Winston, Marie E. Sutherlin, Amanda J. Wright, Evan H. Feinberg, Craig P. Hunter. Caenorhabditis elegans SID-2 is required for environmental RNA interference. Proceedings of the National Academy of Sciences. 2007;104(25):10565-10570. doi:10.1073/pnas.0611282104. - Hiroaki Tabara, Alla Grishok, Craig C. Mello. RNAi in C. elegans : Soaking in the Genome Sequence. Science. 1998;282(5388):430-431. doi:10.1126/science.282.5388.430.

Here we report the identification and characterization of SID-2, an intestinal luminal transmembrane protein required for environmental RNAi in C. elegans.… In plants, this signal is known to be a form of dsRNA, and the signal is most likely a form of dsRNA in C. elegans as well. Furthermore, in C. elegans, dsRNA present in the environment or expressed in ingested bacteria is sufficient to trigger RNAi (environmental RNAi). Ingestion and soaking delivery of dsRNA has also been described for other invertebrates. Here we report the identification and characterization of SID-2, an intestinal luminal transmembrane protein required for environmental RNAi in C. elegans. SID-2, when expressed in the environmental RNAi defective species Caenorhabditis briggsae, confers environmental RNAi. Authors

This dsRNA uptake mechanism seems to be evolutionarily conserved, as knockdown of orthologues in Caenorhabditis elegans inactivated the RNA interference response in worms. … Transport of dsRNA into cells by the transmembrane protein SID-1.… Here, we identify the pathway for dsRNA uptake in Drosophila melanogaster S2 cells. Biochemical and cell biological analyses, and a genome-wide screen for components of the dsRNA-uptake machinery, indicated that dsRNA is taken up by an active process involving receptor-mediated endocytosis. Pharmacological inhibition of endocytic pathways disrupted exogenous dsRNA entry and the induction of gene silencing. This dsRNA uptake mechanism seems to be evolutionarily conserved, as knockdown of orthologues in Caenorhabditis elegans inactivated the RNA interference response in worms. Thus, this entry pathway is required for systemic RNA silencing in whole organisms. In Drosophila cells, pharmacological evidence suggests that dsRNA entry is mediated by pattern-recognition receptors. The possible role of these receptors in dsRNA entry may link RNA interference (RNAi) silencing to other innate immune responses. Conflict of interest statement … Ulvila J, Parikka M, Kleino A, Sormunen R, Ezekowitz RA, Kocks C, Rämet M. Ulvila J, et al. J Biol Chem. 2006 May 19;281(20):14370-5. doi: 10.1074/jbc.M513868200. Epub 2006 Mar 10. J Biol Chem. 2006. PMID: 16531407 Transport of dsRNA into cells by the transmembrane protein SID-1. Feinberg EH, Hunter CP. Feinberg EH, et al. Science. 2003 Sep 12;301(5639):1545-7. doi: 10.1126/science.1087117. Science. 2003. PMID: 12970568

In the nematode Caenorhabditis elegans, a transmembrane protein named systemic RNAi deficient-2 (sid-2) is required for dsRNA uptake from the environment through the intestine.Abstract dsRNA uptake from the environment is a common phenomenon in many invertebrates and can be harnessed for acquiring pest control. In the nematode Caenorhabditis elegans, a transmembrane protein named systemic RNAi deficient-2 (sid-2) is required for dsRNA uptake from the environment through the intestine. Furthermore, the tyrosine-protein kinase sid-3 promotes import dsRNA. However, the molecular mechanism underlying environmental RNA uptake and its natural phenotypic roles are largely unknown. Here I characterized the intracellular properties of SID-2 and its role during the nematode development. I determined SID-2 localization at the intestinal apical membrane and the trans-Golgi-network (TGN). …

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. … Analysis of these mutants suggests that dsRNA uptake is a specific process involving several unique proteins.Abstract 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.

William M. Winston, Marie E. Sutherlin, Amanda J. Wright, Evan H. Feinberg, Craig P. Hunter. Caenorhabditis elegans SID-2 is required for environmental RNA interference. Proceedings of the National Academy of Sciences. 2007;104(25):10565-10570.… V. Hill, Julia M. Claggett, Kate Hill‐Harfe, Joanne C. Wen, Donna Karen Slonim, et al. The homeodomain protein PAL-1 specifies a lineage-specific regulatory network in theC. elegansembryo. Development. 2005;132(8):1843-1854. doi:10.1242/dev.01782. - Lin Liu, Istvan Botos, Yan Wang, Joshua N. Leonard, Joseph Shiloach, David M. Segal, et al. Structural Basis of Toll-Like Receptor 3 Signaling with Double-Stranded RNA. Science. 2008;320(5874):379-381. doi:10.1126/science.1155406. - William M. Winston, Marie E. Sutherlin, Amanda J. Wright, Evan H. Feinberg, Craig P. Hunter. Caenorhabditis elegans SID-2 is required for environmental RNA interference. Proceedings of the National Academy of Sciences. 2007;104(25):10565-10570. doi:10.1073/pnas.0611282104. - Christian Wolfrum, Shuanping Shi, K. Narayanannair Jayaprakash, Muthusamy Jayaraman, Gang Wang, Rajendra K. Pandey, et al. Mechanisms and optimization of in vivo delivery of lipophilic siRNAs. Nature Biotechnology. 2007;25(10):1149-1157. doi:10.1038/nbt1339. - Hiroaki Tabara, Alla Grishok, Craig C. Mello. RNAi in C. elegans : Soaking in the Genome Sequence. Science. 1998;282(5388):430-431. doi:10.1126/science.282.5388.430.