lin-4 and the NRDE pathway are required to activate a transgenic lin-4 reporter but not the endogenous lin-4 locus in C. elegans

Jiao, Alan L.Foster, DanielDixon, JuliaSlack, Frank2018-02-262018Jiao, Alan L., Daniel J. Foster, Julia Dixon, and Frank J. Slack. 2018. “lin-4 and the NRDE pathway are required to activate a transgenic lin-4 reporter but not the endogenous lin-4 locus in C. elegans.” PLoS ONE 13 (1): e0190766. doi:10.1371/journal.pone.0190766. http://dx.doi.org/10.1371/journal.pone.0190766.http://nrs.harvard.edu/urn-3:HUL.InstRepos:34869005As the founding member of the microRNA (miRNA) gene family, insights into lin-4 regulation and function have laid a conceptual foundation for countless miRNA-related studies that followed. We previously showed that a transcriptional lin-4 reporter in C. elegans was positively regulated by a lin-4-complementary element (LCE), and by lin-4 itself. In this study, we sought to (1) identify additional factors required for lin-4 reporter expression, and (2) validate the endogenous relevance of a potential positive autoregulatory mechanism of lin-4 expression. We report that all four core nuclear RNAi factors (nrde-1, nrde-2, nrde-3 and nrde-4), positively regulate lin-4 reporter expression. In contrast, endogenous lin-4 levels were largely unaffected in nrde-2;nrde-3 mutants. Further, an endogenous LCE deletion generated by CRISPR-Cas9 revealed that the LCE was also not necessary for the activity of the endogenous lin-4 promoter. Finally, mutations in mature lin-4 did not reduce primary lin-4 transcript levels. Taken together, these data indicate that under growth conditions that reveal effects at the transgenic locus, a direct, positive autoregulatory mechanism of lin-4 expression does not occur in the context of the endogenous lin-4 locus.en-USBiology and life sciencesGeneticsGene expressionGene regulationMicroRNAsBiochemistryNucleic acidsRNANon-coding RNAEpigeneticsRNA interferenceGenetic interferenceBiology and Life SciencesComputational BiologyGenome ComplexityIntronsGenomicsExperimental Organism SystemsModel OrganismsCaenorhabditis ElegansAnimal ModelsOrganismsEukaryotaAnimalsInvertebratesNematodaCaenorhabditisGene ExpressionGene RegulationCell BiologyCell ProcessesCell Cycle and Cell DivisionAntisense RNAlin-4 and the NRDE pathway are required to activate a transgenic lin-4 reporter but not the endogenous lin-4 locus in C. elegansJournal Article2018-02-2610.1371/journal.pone.0190766