Genetic Modifiers of dFMR1 Encode RNA Granule Components in Drosophila

Mechanisms of neuronal mRNA localization and translation are of considerable biological interest. Spatially regulated mRNA translation contributes to cell-fate decisions and axon guidance during development, as well as to long-term synaptic plasticity in adulthood. The Fragile-X Mental Retardation protein (FMRP/dFMR1) is one of the best-studied neuronal translational control molecules and here we describe the identification and early characterization of proteins likely to function in the dFMR1 pathway. Induction of the dFMR1 in sevenless-expressing cells of the Drosophila eye causes a disorganized (rough) eye through a mechanism that requires residues necessary for dFMR1/FMRP''s translational repressor function. Several mutations in dco, orb2, pAbp, rm62, and smD3 genes dominantly suppress the sev-dfmr1 rough-eye phenotype, suggesting that they are required for dFMR1-mediated processes. The encoded proteins localize to dFMR1-containing neuronal mRNPs in neurites of cultured neurons, and/or have an effect on dendritic branching predicted for bona fide neuronal translational repressors. Genetic mosaic analyses indicate that dco, orb2, rm62, smD3, and dfmr1 are dispensable for translational repression of hid, a microRNA target gene, known to be repressed in wing discs by the bantam miRNA. Thus, the encoded proteins may function as miRNA- and/or mRNA-specific translational regulators in vivo.THE subcellular localization and regulated translation of stored mRNAs contributes to cellular asymmetry and subcellular specialization (Lecuyer et al. 2007; Martin and Ephrussi 2009). In mature neurons, local protein synthesis at active synapses may contribute to synapse-specific plasticity that underlies persistent forms of memory (Casadio et al. 1999; Ashraf et al. 2006; Sutton and Schuman 2006; Richter and Klann 2009). During this process, synaptic activity causes local translation of mRNAs normally stored in translationally repressed synaptic mRNPs (Sutton and Schuman 2006; Richter and Klann 2009). While specific neuronal translational repressors and microRNAs have been implicated in this process, their involvement in local translation that underlies memory, as well as the underlying mechanisms, are generally not well understood (Schratt et al. 2006; Keleman et al. 2007; Kwak et al. 2008; Li et al. 2008; Richter and Klann 2009). Furthermore, it remains possible that there are neuron-specific, mRNA-specific, and stimulus-pattern specific pathways for neuronal translational control (Raab-Graham et al. 2006; Giorgi et al. 2007).The Fragile-X Mental Retardation protein (FMRP) is among the best studied of neuronal translational repressors, in part due to its association with human neurodevelopmental disease (Pieretti et al. 1991; Mazroui et al. 2002; Gao 2008). Consistent with function in synaptic translation required for memory formation, mutations in FMRP are associated with increased synaptic translation, enhanced LTD, increased synapse growth, and also with enhanced long-term memory (Zhang et al. 2001; Huber et al. 2002; Bolduc et al. 2008; Dictenberg et al. 2008).FMRP co-immunoprecipitates with components of the RNAi and miRNA machinery and appears to be required for aspects of miRNA function in neurons (Caudy et al. 2002; Ishizuka et al. 2002; Jin et al. 2004b; Gao 2008). In addition, FMRP associates with neuronal polyribosomes as well as with Staufen-containing ribonucleoprotein (mRNP) granules easily observed in neurites of cultured neurons (Feng et al. 1997; Krichevsky and Kosik 2001; Mazroui et al. 2002; Kanai et al. 2004; Barbee et al. 2006; Bramham and Wells 2007; Bassell and Warren 2008; Dictenberg et al. 2008). FMRP-containing neuronal mRNPs contain not only several ubiquitous translational control molecules, but also CaMKII and Arc mRNAs, whose translation is locally controlled at synapses (Rook et al. 2000; Krichevsky and Kosik 2001; Kanai et al. 2004; Barbee et al. 2006). Thus, FMRP-containing RNA particles are probably translationally repressed and transported along microtubules from the neuronal cell body to synaptic sites in dendrites where local synaptic activity can induce their translation (Kiebler and Bassell 2006; Dictenberg et al. 2008).The functions of FMRP/dFMR1 in mRNA localization as well as miRNA-dependent and independent forms of translational control is likely to require several other regulatory proteins. To identify such proteins, we used a previously designed and validated genetic screen (Wan et al. 2000; Jin et al. 2004a; Zarnescu et al. 2005). The overexpression of dFMR1 in the fly eye causes a “rough-eye” phenotype through a mechanism that requires (a) key residues in dFMR1 that mediate translational repression in vitro; (b) Ago1, a known components of the miRNA pathway; and (c) a DEAD-box helicase called Me31B, which is a highly conserved protein from yeast (Dhh1p) to humans (Rck54/DDX6) functioning in translational repression and present on neuritic mRNPs (Wan et al. 2000; Laggerbauer et al. 2001; Jin et al. 2004a; Coller and Parker 2005; Barbee et al. 2006; Chu and Rana 2006). To identify other Me31B-like translational repressors and neuronal granule components, we screened mutations in 43 candidate proteins for their ability to modify dFMR1 induced rough-eye phenotype. We describe the results of this genetic screen and follow up experiments to address the potential cellular functions of five genes identified as suppressors of sev-dfmr1.