SID-1 Domains Important for dsRNA Import in<i>Caenorhabditis elegans</i>

Whangbo, Jennifer; Weisman, Alexandra S.; Chae, Jeiwook; Hunter, Craig P.

doi:10.1534/g3.117.300308

Cited by 40 publications

(44 citation statements)

References 31 publications

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“…The original large visual screen identified many strong alleles in sid-1 and sid-2 and relatively few weak alleles in sid-3, sid-4 and sid-5. This bias likely reflects the relative ease of identifying worms with bright GFP in many cells in the strong mutants (sid-1 and sid-2 mutants) versus the variable and relatively sparse GFP expression in the weak mutants (sid-3, sid-4, and sid-5, as well as a partial loss-of-function sid-1 alleles, (WHANGBO et al 2017). Our screen and analysis of candidate SID-3/4 interacting proteins extends this pattern, suggesting that most or all strong Sid mutants have been identified.…”

Section: Discussionmentioning

confidence: 99%

“…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).…”

Section: Introductionmentioning

confidence: 99%

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

Bhatia

2019

Preprint

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RNA interference (RNAi) is sequence-specific gene silencing triggered by double-stranded (ds)RNA. When dsRNA is expressed or introduced into one cell and is transported to and initiates RNAi in other cells, it is called systemic RNAi. Systemic RNAi is very efficient in C. elegans and genetic screens for systemic RNAi defective (Sid) 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, dosesensitive, systemic RNAi defect and can be effectively rescued by SID-4 expression in target tissues only, implying a role in dsRNA import. SID-4 and SID-3 (ACK-1 kinase) homologs interact in mammals and insects, suggesting they may function in a common signaling pathway, however, a sid-3; sid-4 double mutants showed additive resistance to RNAi, suggesting that these proteins likely interact with other signaling pathways as well. A bioinformatic screen coupled to RNAi sensitivity tests identified 23 additional signaling components with weak RNAi defective phenotypes. These observations suggest that environmental conditions may modulate systemic RNAi efficacy, and indeed, sid-3 and sid-4 are required for growth temperature effects on systemic RNAi silencing efficiency.* Partial Dpy's, Supplemental Figures Figure S1. Lineal relatedness of cells that fuse to form syncytial hypodermal cell hyp7.A) Lineage tree showing the cell divisions (vertical lines) leading to the seven early blastomeres that give rise to embryonic (E) or postembryonic (PE) cells that fuse with hyp7. The blue bar show the relative contributions to hyp7 and the green circles show the origins of pharyngeal muscle cells that express sid-4::gfp. B, C) Examples of extrachromosomal array transmission patterns that could generate the patterns observed in bli-1 (RNAi) resistant sid-4::gfp positive mosaic animals ( Figure 6). B) At the first division the array segregates to P1 but not to AB and at the second division segregates to EMS but not to P2. The resulting animal expresses sid-4::gfp in the intestine and cells of the posterior pharynx, but lacks sid-4::gfp in all hyp7 nuclei. C) At the first division the array segregates to AB but not to P1, at the second division segregates to ABa but not to ABp, and at a subsequent division segregates to ABar, ABarp, or ABarpp. The resulting animal expresses sid-4::gfp in cells of the anterior pharynx, but lacks sid-4::gfp expression in all or most hpy7 nuclei.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Bhatia

2019

Preprint

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“…The gene product of the down-regulated SID1 Transmembrane Family Member 1 (SIDT1) is involved in RNA-interference (RNAi), by transporting dsRNA across cellular membranes (e.g., Whangbo et al, 2017). The down-regulation suggests that gene silencing of RNAi inhibited genes are being removed, which allows transcription.…”

Section: Discussionmentioning

confidence: 99%

Environmental Stress Responses and Experimental Handling Artifacts of a Model Organism, the Copepod Acartia tonsa (Dana)

et al. 2018

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Handling animals during experiments potentially affects the differential expression of genes chosen as biomarkers of sub-lethal stress. RNA sequencing was used to examine whole-transcriptome responses caused by laboratory handling of the calanoid copepod, Acartia tonsa. Salinity shock (S = 35 to S = 5) was used as positive stress control; individuals not exposed to handling or other stressors served as negative stress control. All copepods were grown from eggs to adults without being handled or exposed to any stressors prior the experiment. Survival of nauplii and adults was estimated for up to 10 min of exposure to handling stress and salinity shock. Only adults exhibited decreased survival (44 ± 7% with 10 min of exposure) in response to handling stress and were selected for definitive experiments for RNA sequencing. After 10 min of experimental exposures to handling stress or salinity shock, adults were incubated for 15 min or 24 h at normal culture conditions. A small number of significantly differentially expressed genes (DEGs) were observed 15 min after exposure to handling stress (2 DEGs) or salinity shock (7 DEGs). However, 24 h after exposure, handling stress resulted in 276 DEGs and salinity shock resulted in 573 DEGs, of which 174 DEGs were overlapping between the treatments. Among the DEGs observed 24 h after exposure to handling stress or salinity shock, some commonly-used stress biomarkers appeared at low levels. This suggests that a stress-response was induced at the transcriptional level for these genes between 15 min and 24 h following exposure. Since handling stress clearly affects transcriptional patterns, it is important to consider handling when designing experiments, by either including additional controls or avoiding focus on impacted genes. Not considering handling in gene expression studies can lead to inaccurate conclusions. The present study provides a baseline for studying handling stress in future studies using this model organism and others.

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“…The amino acids are colored by similarity. Several residues that were identified by Whangbo et al (Whangbo et al, 2017) as being important for the C. elegans SID-1 activity are boxed in red. The alignment shows that most these residues are not conserved with CHUP-1 or with the insect SIL proteins.…”

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confidence: 99%

“…is necessary for cellular uptake of environmental cholesterol (Valdes et al, 2012), and does not support dsRNA uptake when heterologously expressed in Drosophila S2 cells (Whangbo et al, 2017). Other mammalian SID-related proteins, SIDT1 and SIDT2, were also shown to bind and influence cholesterol transport (SIDT1 at the plasma membrane and SIDT2 within the cell) but not dsRNA or miRNA (Mendez-Acevedo et al, 2017).…”

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confidence: 99%

The mysteries of insect RNAi: A focus on dsRNA uptake and transport

Vélez

Fishilevich

2018

Pesticide Biochemistry and Physiology

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RNA interference (RNAi) is becoming a practical tool to control insect pests. Many mysteries of how double-stranded RNA (dsRNA) is transported into, within, and between cells to generate an efficient RNAi response in insects are still to be unraveled. This review provides an overview of the evidence that supports a key role of endocytosis in the uptake of dsRNA on both cellular and tissue levels. Additionally, other components of cellular membrane transport and their impact on the efficiency of RNAi in insects are explored. It is now evident that the membrane transport and potentially dsRNA release from the endosome may comprise some of the limiting factors in insects that are recalcitrant to dsRNA. This review concludes with the apparent connection between gene products that are necessary for cellular trafficking of dsRNA and highly lethal RNAi targets.

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SID-1 Domains Important for dsRNA Import inCaenorhabditis elegans

Cited by 40 publications

References 31 publications

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

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

Environmental Stress Responses and Experimental Handling Artifacts of a Model Organism, the Copepod Acartia tonsa (Dana)

The mysteries of insect RNAi: A focus on dsRNA uptake and transport

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