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Abstract

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.