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Landscape and functions of RNA localization in the Drosophila follicular epithelium
The mechanisms and roles underlying the phenomenon of RNA localization in differentiated tissues are poorly characterized. Drosophila follicular epithelium represents a good model to study RNA localization in the context of an adult tissue, for several reasons. First, it expresses the dynein/BicD/Egl RNA localization machinery, which transports RNAs to the apical domain. Second, it is genetically tractable, offering the possibility to generate mutant clones for a gene of interest in a mosaic manner. So far, only a handful of mRNAs showing a subcellular localization pattern in the follicular epithelium have been reported, and the extent and significance of the phenomenon are not known. To address this, I applied a spatial transcriptomics approach to identify the mRNAs that show apical or basal localization patterns in this tissue. Laser-capture microdissection (LCM) followed by RNA-seq and bioinformatic analysis allowed the identification of 520 differentially enriched mRNAs in either the apical or the basal domain. Gene Ontology (GO) enrichment analysis showed a functional compartmentalization of mRNAs in different cellular domains, suggesting that mRNAs encoding components of the same organelle or pathway might be locally translated. Next, I investigated the mechanisms that govern apical and basal RNA localization in the follicular epithelium, by knocking down components of known microtubule (MT) minus end- and plus end-directed RNA transport. I found that apical RNA localization is achieved mainly by the activity of the dynein/BicD/Egl RNA transport machinery, but that at least one mRNA is localized apically by another MT-based mechanism. On the other hand, basal RNA localization is dependent on kinesin-1 and, in some cases, is regulated by the deposition of the Exon Junction Complex (EJC), reminiscent of the mechanisms that underlie oskar RNA localization to the posterior pole. Interestingly, disrupting kinesin-1 by Khc RNAi causes basal mRNAs to localize apically. I have shown that such apical mislocalization of a basal RNA depends on egalitarian (egl), but that the basal RNA’s localization is not affected upon sole knock-down of egl. Based on this, I propose a model whereby the localization of basal mRNAs is accomplished by kinesin-1, which counteracts a default apical RNA transport pathway by inhibiting the activity of the dynein/BicD/Egl machinery. Finally, I investigated the role of Egl in apical localization of Imp mRNA by analyzing eglRNAi follicle cell clones and measuring the hatching rate of eggs generated from egg-chambers subjected to egl RNAi in the follicle cells. However, absence of Egl in the follicle cells caused only a small reduction in egg hatching rate. It is possible that egalitarian-dependent apical localization of mRNAs in the follicular epithelium becomes important in conditions of stress encountered by fruit flies in a natural environment.
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570 Biowissenschaften, Biologie
570 Life sciences