Biology subjects

Hunter, C. P.

Publications and source records attributed to Hunter, C. P..

5 recordsLinked to original sources

bioRxiv2024

Evidence for in vivo mRNA Transport Between Mammalian Cells

The prevalence and significance of intercellular mRNA transport remains unknown. Direct detection of mRNA transfer between cells within an organism is challenging due to technical limitations associated with transgene encoded molecular labels and cell sorting techniques. In this study we analyzed human-to-mouse xenograft single-cell RNA sequencing data to identify mouse transcripts in recovered human cells. The murine transcriptome analysis implicates macrophages as a frequent mRNA donor cell type. We then developed an in vitro system using mouse RAW264.7 macrophages and human HeLa and HEK293 cells and confirmed the transfer of Ftl1 mRNA from mouse to human cells. Overall, our study provides compelling in vivo evidence for prevalent intercellular mRNA transfer in human-to-mouse xenograft models.

cell biology↗

bioRxiv2024

Irreproducibility of transgenerational learned pathogen-aversion response in C. elegans

Here we report our attempt to replicate reports of transgenerational epigenetic inheritance in Caenorhabditis elegans. Published results from multiple laboratories show that C. elegans adults and their F1 embryos exposed to the pathogen Pseudomonas aeruginosa show pathogen aversion behavior and a pathogen exposure-dependent increase in daf-7/TGF{beta} reporter gene expression. However, results from one group show persistence of the aversion behavior and elevated daf-7 expression in the F2, F3, and F4 generations. In contrast, we failed to consistently detect either the pathogen avoidance response or elevated daf-7 expression beyond the F1 generation. We did confirm that the dsRNA transport proteins SID-1 and SID-2 are required for the intergenerational (F1) inheritance of pathogen avoidance, but not for the F1 inheritance of elevated daf-7 expression. Furthermore, our reanalysis of RNA seq data provides additional evidence that this intergenerational inherited PA14 response may be mediated by small RNAs. The experimental methods are well-described, the source materials are readily available, including samples from the reporting laboratory, and we explored a variety of environmental conditions likely to account for lab-to-lab variability. None of these adjustments altered our results. We conclude that this example of transgenerational inheritance lacks robustness, confirm that the intergenerational avoidance response, but not the elevated daf-7p::gfp expression in F1 progeny, requires sid-1 and sid-2, and identify candidate siRNAs and target genes that may mediate this intergenerational response.

genetics↗

bioRxiv2019

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

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.

genetics↗

bioRxiv2017

SID-1 domains important for dsRNA import in C. 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 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.

genetics↗

bioRxiv2017

Characterization of SID-1-dependent and independent intergenerational RNA transport pathways in Caenorhabditis elegans

Systemic RNA interference (RNAi) in C. elegans is dependent on sid-1 (WO_SCPLOWINSTONC_SCPLOW et al. 2002), sid-3 (JO_SCPLOWOSEC_SCPLOW et al. 2012) and sid-5 (HO_SCPLOWINASC_SCPLOW et al. 2012). After injection, expression, or ingestion, double-stranded RNA (dsRNA) is transported between cells throughout the animal to enable RNAi in most tissues, including the germline and progeny. Here, we characterize the role of the Sid genes in transport of dsRNA to progeny. We previously reported that dsRNA injected directly in the germline unexpectedly requires sid-1 activity in the progeny to initiate RNAi (WO_SCPLOWINSTONC_SCPLOW et al. 2002). We now show that germline injected dsRNA can travel by three independent pathways to silence gene expression in embryos. First, germline injected dsRNA is delivered, presumably by bulk flow, into oocytes and embryos. This means of delivery, which does not require sid-1, is limited by the amount and location of injected dsRNA. Second, maternal sid-1 transports extracellular dsRNA into the germline where it can silence maternal deposited mRNAs and segregate to embryos to silence embryonically expressed mRNAs. Third, extracellular dsRNA is also endocytosed into oocytes by the low-density lipoprotein (LDL) receptor superfamily homolog RME-2. The endocytosed dsRNA then requires sid-1 and sid-5 in embryos to silence embryonically expressed genes. Extracellular fluorescent dsRNA, once endocytosed into oocytes, does not co-localize with VIT2::GFP and it does not require sid-1 activity to segregate from the late endocytosis marker GFP::RAB-7 in early embryos. In conclusion, we identify genes and pathways that function redundantly for intergenerational RNA transfer that may represent mechanisms for transgenerational epigenetic inheritance.

genetics↗