Spatiotemporal control of protein expression is essential for neuronal development and plasticity. Fragile X mental retardation protein (FMRP) is an RNA-binding protein involved in regulated translation of numerous specific target mRNAs in neurons, although the mechanisms by which FMRP regulates translation are not well defined. The RNA-induced silencing complex (RISC) is also an important mediator of translational control and recent evidence indicates that FMRP recruits RISC to cooperatively regulate stimulus-dependent translation of the mRNA encoding the synaptic protein PSD-95. However, it is unknown how widely the mechanism of RISC recruitment by FMRP is used for FMRP-dependent translational regulation in neurons. Based on the working hypothesis that FMRP broadly regulates the association of specific miRNAs with the RISC complex to modulate translation at the synapse, the following two aims are proposed: 1) Identify specific miRNAs that facilitate the FMRP-dependent stimulus-induced translation of an important FMRP target, GluA1 mRNA;and 2) Assess the extent to which Fmr1-/- alters the association of the RISC protein Ago2 with specific miRNAs at the synapse.
These aims will be accomplished using primary cortical neuron culture, RNA immunoprecipitation, RNA-FISH and immunofluorescence microscopy, qRT-PCR, and miRNA sequencing, and will require training in neuroscience, microscopy, and bioinformatics. These experiments will illuminate a novel mechanism by which FMRP bi-directionally regulates translation with fine spatial and temporal control. Understanding the mechanisms underlying FMRP-dependent translational control will provide fundamental insight into the regulated protein expression involved in normal brain development and shed light on neurological problems such as intellectual disability, neurodegeneration, epilepsy, and autism.
Intellectual disability, autism, and epilepsy are all associated with disruption of FMRP, which participates in controlling the timing and location of protein production that is important for proper brain development and function. The RISC complex is also important for controlling protein production, and the proposed studies will address how FMRP affects the RISC components Ago2, miRNA, and mRNA. Understanding how FMRP alters the RISC complex will give insight into normal and abnormal brain development, allowing us to better understand neurological problems and FMRP-associated disorders.
|Suhl, Joshua A; Chopra, Pankaj; Anderson, Bart R et al. (2014) Analysis of FMRP mRNA target datasets reveals highly associated mRNAs mediated by G-quadruplex structures formed via clustered WGGA sequences. Hum Mol Genet 23:5479-91|