Post-transcriptional gene regulation in Drosophila : an investigation into the roles of RNA silencing and the DEAD-box helicase Belle

Post-transcriptional regulation of gene expression relies on multiple mechanisms to elicit translational control and/or mRNA decay. RNA silencing pathways operate at both stages, targeting a significant fraction of the transcriptome, namely those mRNAs complementary to short interfering RNAs (siRNAs) and microRNAs (miRNAs). Using Drosophila S2 cells, I have carried out a genome-wide search for transcripts regulated by these pathways. mRNA expression profiles were obtained for cells depleted of AGO1, AGO2, PIWI or Aubergine, members of the Argonaute family of proteins essential for RNA silencing, and analyzed alongside profiles from cells depleted of the miRNA-processing enzyme Drosha. Changes in transcript levels in Drosha-depleted cells correlated closely with those in the AGO1 knockdown, demonstrating that miRNA targets change level following inhibition of the miRNA pathway and supporting the idea that miRNAs can cause degradation of the targeted transcripts, and do not just repress translation as previously thought. Furthermore, it was found that a subset of miRNA targets is also regulated by AGO2; together with evidence that AGO1 and AGO2 silence the expression of a common set of mobile genetic elements, this suggests a degree of functional overlap for AGO1 and AGO2 in the Drosophila RNA silencing pathway. I next focused on the Drosophila protein Belle, a member of the conserved family of DEAD-box RNA helicases. Most members of this protein family exhibit NTPase activity stimulated by or dependent on RNA binding and use the energy derived from NTP hydrolysis to unwind double-stranded RNA or disrupt RNA/protein interactions. Many DEAD-box proteins localize to RNA granules such as maternal and neuronal transport mRNPs, polar granules, and P bodies. Belle is a component of nuage within nurse cells and polar granules within the oocyte. It is an essential protein required for larval growth, as well as male and female fertility, and has a putative role in RNA silencing. I show that Belle is required for cell viability. In cells depleted of Belle, general protein synthesis is inhibited. However, a luciferase mRNA reporter with Belle tethered to the 3′ UTR is translationally repressed without any reduction in mRNA levels. Tethering of Belle to the reporter mRNA induces the formation of heavy mRNP complexes. Translational repression is abolished when Belle contains mutations disrupting the putative helicase activity. Using a biochemical and computational approaches, I found that in Drosophila S2 cells, Belle is part of an interaction network consisting of proteins implicated in general protein synthesis as well as selective translational control mechanisms. Belle interacts with translation initiation factors and ribosomal proteins, supporting the idea that Belle is required for general translation efficiency. However it also associates with translationally regulated mRNAs and proteins involved in mRNA translational control and localization, suggesting that Belle may be implicated in mRNP assembly and transport as well as localized mRNA translation. Finally, I show that Belle interacts with AGO2 and other components of the RISC, suggesting that Belle may function as an auxiliary factor in the RNA silencing pathway.