Characterization of Fragile X Mental Retardation Protein Recruitment and Dynamics in Drosophila Stress Granules

Abstract

The RNA-binding protein Fragile X Mental Retardation (FMRP) is an evolutionarily conserved protein that is particularly abundant in the brain due to its high expression in neurons. FMRP deficiency causes fragile X mental retardation syndrome. In neurons, FMRP controls the translation of target mRNAs in part by promoting dynamic transport in and out neuronal RNA granules. We and others have previously shown that upon stress, mammalian FMRP dissociates from translating polysomes to localize into neuronal-like granules termed stress granules (SG). This localization of FMRP in SG is conserved in Drosophila. Whether FMRP plays a key role in SG formation, how FMRP is recruited into SG, and whether its association with SG is dynamic are currently unknown. In contrast with mammalian FMRP, which has two paralog proteins, Drosophila FMR1 (dFMRP) is encoded by a single gene that has no paralog. Using this genetically simple model, we assessed the role of dFMRP in SG formation and defined the determinants required for its recruitment in SG as well as its dynamics in SG. We show that dFMRP is dispensable for SG formation in vitro and ex vivo. FRAP experiments showed that dFMRP shuttles in and out SG. The shuttling activity of dFMRP is mediated by a protein-protein interaction domain located at the N-terminus of the protein. This domain is, however, dispensable for the localization of dFMRP in SG. This localization of dFMRP in SG requires the KH and RGG motifs which are known to mediate RNA binding, as well as the C-terminal glutamine/asparagine rich domain. Our studies thus suggest that the mechanisms controlling the recruitment of FMRP into SG and those that promote its shuttling between granules and the cytosol are uncoupled. To our knowledge, this is the first demonstration of the regulated shuttling activity of a SG component between RNA granules and the cytosol.

FMRP and Ataxin-2 function together in long-term olfactory habituation and neuronal translational control

“…The observations above are consistent with recent independent experiments indicating that dFMR1 is not required for the assembly of stress granules in Drosophila nonneuronal cells (64). Taken together, the data indicate that although dFmr1 and Atx2 function together with Me31B in the translational regulation of the dendritic CaMKII reporter mRNA, dFmr1 is dispensable but Atx2 broadly necessary for the presence of Me31B foci that represent likely sites of RNA regulation in neurons.…”

“…This would suggest that Atx2 contains one or more functional domains missing in dFmr1 that allow the multivalent interactions necessary for mRNP assembly. This is most consistent with the observation that that although dFMR1 is a component of stress granules in Drosophila nonneuronal cells, it is not required for their assembly (64). An alternative model would allow both dFmr1 and Atx2 to mediate mRNP assembly but posit that dFmr1 is only present on a small subset of mRNPs, in contrast to Atx2, which is present on the majority.…”

Arginine methylation augments Sbp1 function in translation repression and decapping

“…It is possible that Sbp1 directly modulates decapping activity by binding decapping complex or mRNA or both. Consistent with its role in repression and decapping, Sbp1 localizes to RNA granules [9] akin to other translation repressors such as FMRP, CUP, Ded1 and Pat1 [10][11][12][13]. Co-localization of Sbp1 foci with enhancer of mRNA decapping (Edc3) foci (PB marker) is slightly more than with Pub1 (SG marker) foci [9] but the significance of this observation is unclear.…”

Analysis of Translation Initiation During Stress Conditions by Polysome Profiling

“…Note that treatment with the proteasome inhibitor reduces polysomes peaks with a concomitant increases in 80S monosomes indicating an inhibition of translation initiation. Typical profiles obtained from Schneider Drosophila cells or mice brain have been described elsewhere [7][8][9][10] . Click here to view larger image.…”