Long noncoding RNAs (lncRNAs) constitute the majority of transcripts in the mammalian genomes, and yet, their functions remain largely unknown. As part of the FANTOM6 project, we systematically knocked down the expression of 285 lncRNAs in human dermal fibroblasts and quantified cellular growth, morphological changes, and transcriptomic responses using Capped Analysis of Gene Expression (CAGE). Antisense oligonucleotides targeting the same lncRNAs exhibited global concordance, and the molecular phenotype, measured by CAGE, recapitulated the observed cellular phenotypes while providing additional insights on the affected genes and pathways. Here, we disseminate the largest-todate lncRNA knockdown data set with molecular phenotyping (over 1000 CAGE deep-sequencing libraries) for further exploration and highlight functional roles for ZNF213-AS1 and lnc-KHDC3L-2.
The intestine is a common site for invasion by intracellular pathogens, but little is known about how pathogens restructure and exit intestinal cells in vivo. The natural microsporidian parasite N. parisii invades intestinal cells of the nematode C. elegans, progresses through its life cycle, and then exits cells in a transmissible spore form. Here we show that N. parisii causes rearrangements of host actin inside intestinal cells as part of a novel parasite exit strategy. First, we show that N. parisii infection causes ectopic localization of the normally apical-restricted actin to the basolateral side of intestinal cells, where it often forms network-like structures. Soon after this actin relocalization, we find that gaps appear in the terminal web, a conserved cytoskeletal structure that could present a barrier to exit. Reducing actin expression creates terminal web gaps in the absence of infection, suggesting that infection-induced actin relocalization triggers gap formation. We show that terminal web gaps form at a distinct stage of infection, precisely timed to precede spore exit, and that all contagious animals exhibit gaps. Interestingly, we find that while perturbations in actin can create these gaps, actin is not required for infection progression or spore formation, but actin is required for spore exit. Finally, we show that despite large numbers of spores exiting intestinal cells, this exit does not cause cell lysis. These results provide insight into parasite manipulation of the host cytoskeleton and non-lytic escape from intestinal cells in vivo.
Pathogen exit is a key stage in the spread and propagation of infectious disease, with the fecal-oral route being a common mode of disease transmission. However, it is poorly understood which molecular pathways provide the major modes for intracellular pathogen exit and fecal-oral transmission in vivo. Here, we use the transparent nematode Caenorhabditis elegans to investigate intestinal cell exit and fecal-oral transmission by the natural intracellular pathogen Nematocida parisii, which is a recently identified species of microsporidia. We show that N. parisii exits from polarized host intestinal cells by co-opting the host vesicle trafficking system and escaping into the lumen. Using a genetic screen, we identified components of the host endocytic recycling pathway that are required for N. parisii spore exit via exocytosis. In particular, we show that the small GTPase RAB-11 localizes to apical spores, is required for spore-containing compartments to fuse with the apical plasma membrane, and is required for spore exit. In addition, we find that RAB-11-deficient animals exhibit impaired contagiousness, supporting an in vivo role for this host trafficking factor in microsporidia disease transmission. Altogether, these findings provide an in vivo example of the major mode of exit used by a natural pathogen for disease spread via fecal-oral transmission.
Microsporidia comprise one of the largest groups of obligate intracellular pathogens and can infect virtually all animals, but host response to these fungal-related microbes has been poorly understood. Several new studies of the host transcriptional response to microsporidia infection have found infection-induced regulation of genes involved in innate immunity, ubiquitylation, metabolism, and hormonal signaling. In addition, microsporidia have recently been shown to exploit host recycling endocytosis for exit from intestinal cells, and to interact with host degradation pathways. Microsporidia infection has also been shown to profoundly affect behavior in insect hosts. Altogether, these and other recent findings are providing much-needed insight into the underlying mechanisms of microsporidia interaction with host animals.
Long non-coding RNAs (lncRNAs) constitute the majority of transcripts in the mammalian genomes and yet, their functions remain largely unknown. We systematically knockdown 285 lncRNAs expression in human dermal fibroblasts and quantified cellular growth, morphological changes, and transcriptomic responses using Capped Analysis of Gene Expression (CAGE). Antisense oligonucleotides targeting the same lncRNA exhibited global concordance, and the molecular phenotype, measured by CAGE, recapitulated the observed cellular phenotypes while providing additional insights on the affected genes and pathways. Here, we disseminate the largest to-date lncRNA knockdown dataset with molecular phenotyping (over 1,000 CAGE deep-sequencing libraries) for further exploration and highlight functional roles for ZNF213-AS1 and lnc-KHDC3L-2.
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