490C Poster - 06. Regulation of gene expression
Saturday April 09, 1:30 PM - 3:30 PM

Development of a novel molecular assay to sensitively detect Fmr1's translational function in Drosophila ovarian follicles.


Authors:
Kayla Judson; Al Rohet Hossain; Kaicheng Ma

Affiliation: Department of Biochemistry and Molecular Biology, UBC, Vancouver, BC

Keywords:
h. translational regulation; u. RNA binding proteins

Loss of function mutations in the Fragile X Mental Retardation 1 (Fmr1) gene are associated
with fragile X syndrome and fragile X-associated primary ovarian insufficiency, leading causes of
autism and female infertility respectively. Although Fmr1 has been highly studied, its biological
role is still subject to debate. A major challenge in studying Fmr1 is that its molecular function is
to weakly promote the translation of its targets ~2-fold, making it difficult to assess its activity
in vivo. Here we present a novel assay to detect Fmr1’s function at a molecular level with high
sensitivity in developing Drosophila ovarian follicles.

Using single molecule fluorescence in situ hybridization (smFISH), we observed that the mRNA
of the Fmr1 target Poe forms microscopically visible granules that depend on ongoing
translation. These "Poe particles" appear to be sites of Fmr1 translational activation, as Fmr1,
Poe mRNA, and fully translated Poe protein are each enriched in Poe granules. Fmr
knockdown, or treatments that reduce translation (puromycin or heat shock) resulted in a
robust dispersal in Poe mRNA granules. Using the presence of Poe particles as a sensitive
readout of Fmr1 activity, we conducted a genetic screen to identify potential partners of Fmr1,
by labeling Poe mRNAs in either wild type egg chambers or those lacking Fmr1 or candidate
genes. We screened over a hundred different candidate genes using germline-specific RNAi. We
found that only a few RNAi lines targeting RNA binding proteins and/or proteins which
physically bind to Fmr1 exhibited a strong reduction in Poe particles. Our findings suggest that
Fmr1 functions together with a small set of additional factors to promote the expression of
genes essential for normal ovarian and neuronal function.