Sid Watkins
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PROVIDER: S-EPMC1126417 | biostudies-literature | 2003 Jul
REPOSITORIES: biostudies-literature
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Project description: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.
| S-EPMC3488460 | biostudies-literature
Project description:We expressed SID-1, a transmembrane protein from Caenorhabditis elegans that is required for systemic RNA interference (RNAi), in C. elegans neurons. This expression increased the response of neurons to double-stranded (ds)RNA delivered by feeding. Mutations in the lin-15b and lin-35 genes enhanced this effect. Worms expressing neuronal SID-1 showed RNAi phenotypes when fed with bacteria expressing dsRNA for known neuronal genes and for uncharacterized genes with no previously known neuronal phenotypes. Neuronal expression of sid-1 decreased nonneuronal RNAi, suggesting that neurons expressing transgenic sid-1(+) served as a sink for dsRNA. This effect, or a sid-1(-) background, can be used to uncover neuronal defects for lethal genes. Expression of sid-1(+) from cell-specific promoters in sid-1 mutants results in cell-specific feeding RNAi. We used these strains to identify a role for integrin signaling genes in mechanosensation.
| S-EPMC2894993 | biostudies-literature
Project description:The development of native mass spectrometry (MS) has provided structural biologists an additional tool to probe the structures of large macromolecular systems. Surface-induced dissociation (SID) is one activation method used within tandem MS experiments that has proven useful in interrogating the connectivity and topology of biologically-relevant protein complexes. We present here the use of a tilted surface and ion carpet array within a new SID device design, enabling decreased dimensions along the ion path and fewer lenses to tune. This device works well in fragmenting ions of both low (peptides) and high (protein complexes) m/z. Results show that the ion carpet array, while enabling simplification of the back-end of the device, has deficiencies in product collection and subsequently signal at higher SID energies when fragmenting protein complexes. However, the use of the tilted surface is advantageous as an effective way to shorten the device and reduce the number of independent voltages.
| S-EPMC7203677 | biostudies-literature
Project description:Systemic RNAi in Caenorhabditis elegans requires the widely conserved transmembrane protein SID-1 to transport RNAi silencing signals between cells. When expressed in Drosophila S2 cells, C. elegans SID-1 enables passive dsRNA uptake from the culture medium, suggesting that SID-1 functions as a channel for the transport of double-stranded RNA (dsRNA). Here we show that nucleic acid transport by SID-1 is specific for dsRNA and that addition of dsRNA to SID-1 expressing cells results in changes in membrane conductance, which indicate that SID-1 is a dsRNA gated channel protein. Consistent with passive bidirectional transport, we find that the RNA induced silencing complex (RISC) is required to prevent the export of imported dsRNA and that retention of dsRNA by RISC does not seem to involve processing of retained dsRNA into siRNAs. Finally, we show that mimics of natural molecules that contain both single- and double-stranded dsRNA, such as hairpin RNA and pre-microRNA, can be transported by SID-1. These findings provide insight into the nature of potential endogenous RNA signaling molecules in animals.
| S-EPMC3096038 | biostudies-literature
Project description:In plants and in the nematode Caenorhabditis elegans, an RNAi signal can trigger gene silencing in cells distant from the site where silencing is initiated. 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.
| S-EPMC1965553 | biostudies-literature
Project description:We describe a set of simple devices for surface-induced dissociation of proteins and protein complexes on three instrument platforms. All of the devices use a novel yet simple split lens geometry that is minimally invasive (requiring a few millimeters along the ion path axis) and is easier to operate than prior generations of devices. The split lens is designed to be small enough to replace the entrance lens of a Bruker FT-ICR collision cell, the dynamic range enhancement (DRE) lens of a Waters Q-IM-TOF, or the exit lens of a transfer multipole of a Thermo Scientific Extended Mass Range (EMR) Orbitrap. Despite the decrease in size and reduction in number of electrodes to 3 (from 10 to 12 in Gen 1 and ?6 in Gen 2), we show sensitivity improvement in a variety of cases across all platforms while also maintaining SID capabilities across a wide mass and energy range. The coupling of SID, high resolution, and ion mobility is demonstrated for a variety of protein complexes of varying topologies.
| S-EPMC7438310 | biostudies-literature
Project description:In C. elegans, the cell surface protein Sid-1 imports extracellular dsRNA into the cytosol of most non-neuronal cells, enabling systemic spread of RNA interference (RNAi) throughout the worm. Sid-1 homologs are found in many other animals, although for most a function for the protein has not yet been established. Sid-1 proteins are composed of an N-terminal extracellular domain (ECD) followed by 9-12 predicted transmembrane regions. We developed a baculovirus system to express and purify the ECD of the human Sid-1 protein SidT1. Recombinant SidT1 ECD is glycosylated and spontaneously assembles into a stable and discrete tetrameric structure. Electron microscopy (EM) and small angle x-ray scattering (SAXS) studies reveal that the SidT1 ECD tetramer is a compact, puck-shaped globular particle, which we hypothesize may control access of dsRNA to the transmembrane pore. These characterizations provide inroads towards understanding the mechanism of this unique RNA transport system from structural prospective.
| S-EPMC3324469 | biostudies-literature
Project description:RNA interference (RNAi) is a gene regulatory mechanism based on RNA-RNA interaction conserved through eukaryotes. Surprisingly, many animals can take-up human-made double stranded RNA (dsRNA) from the environment to initiate RNAi suggesting a mechanism for dsRNA-based information exchange between organisms and their environment. However, no naturally occurring example has been identified since the discovery of the phenomenon 22 years ago. Therefore it remains enigmatic why animals are able to take up dsRNA. Here, we explore other possible functions by performing phenotypic studies of dsRNA uptake deficient sid-2 mutants in Caenorhabditis elegans. We find that SID-2 does not have a nutritional role in feeding experiments using genetic sensitized mutants. Furthermore, we use robot assisted imaging to show that sid-2 mutants accelerate growth rate and, by maternal contribution, body length at hatching. Finally, we perform transcriptome and lipidome analysis showing that sid-2 has no effect on energy storage lipids, but affects signalling lipids and the embryo transcriptome. Overall, these results suggest that sid-2 has mild effects on development and is unlikely functioning in the nutritional uptake of dsRNA. These findings broaden our understanding of the biological role of SID-2 and motivate studies identifying the role of environmental dsRNA uptake.
| S-EPMC8081039 | biostudies-literature
Project description:Abstract The sid-1 (systemic interference defective) gene encodes a transmembrane protein that is an important participator in the systemic RNAi pathway and has been reported in several organisms. In insects, sid-1-like genes were described from Tribolium castaneum, Apis mellifera, Bombyx mori and Schistocerca americana, but were not found in Drosophila melanogaster and Anopheles gambiae. To investigate whether this gene occurs in aphid species, RT-PCRs were performed using degenerate primers designed using the conserved motif of sid-1-like genes. An sid-1-like full-length transcript was amplified from the cotton/melon aphid, Aphis gossypii Glover (Homopera: Aphididae), and a fragment was amplified from the grain aphid, Sitobion avenae (F.). The transcript from A. gossypii was 3067 bp long, with an open reading frame encoding 766 amino acids. Sequence analysis indicated that this transcript shares highest similarity with the reported sid-1-like gene in Schistocerca americana (53%, fragment), followed by A. mellifera (44%), T. castaneum (32-44%), B. mori (38-42%) and Caenorhabditis elegans (25%). Analysis of the transmembrane protein topological structure indicated that the protein encoded by this gene has a similar structure to SID-1 of C. elegans. A phylogenetic tree with all available sid-1-like genes suggests that sid-1-like genes may have had a long evolutionary history. Considering its importance in the RNAi pathway, the absence of a sid-1-like gene in D. melanogaster and A. gambiae is worthy of further investigation.
| S-EPMC3061602 | biostudies-literature