Silencing of the Fragile X Mental Retardation Protein (FMRP)-encoding gene Fmr1 causes Fragile X Syndrome, the leading known cause of autism. FMRP is an alternatively spliced, multidomain, RNA-binding protein most highly expressed in the brain that regulates mRNA transport and translation. Its function is well-characterized in dendrites but it is also found in the cell body, axons, and in the nucleus. Functioning throughout neurons suggests that some mechanism exists whereby it is appropriately transported and that this mechanism requires one or more of its domains. FMRP is alternatively spliced to yield at least 12 splice forms. These differ from each other in which domains and post-translational modification sites are present or absent. Our hypothesis is that one or more domains is required for axonal localization of FMRP. This is tested using EGFP-tagged FMRP splice form constructs to identify whether certain ones are preferentially localized to axons over others as well as mutation-containing constructs to identify one or more domains that are required for the mechanism of axonal localization of FMRP. These constructs are transfected into cultured rat cortical neurons with tdTomato and examined for differences in axonal localization, length, puncta distribution and density, and axonal arbor complexity, a process regulated by FMRP. All splice forms showed axonal localization at equivalent efficiencies and quantities suggesting that all are able to function in axons. Since the N-terminus is well-conserved between splice forms while the C-terminus is more variable due to the alternative splicing events, it was concluded that the domain required for axonal localization is N-terminal to the first splice site. Using a model for FMRP regulation of axonal arbor complexity in which overexpression of splice forms that function in axon growth and branching would oversimplify axonal arbors, SF7 and SF9-transfected neurons had reduced complexity. The domain(s) required for this function may be C-terminal. Because there are several domains in this region affected by alternative splicing, individual mutant analysis was required to narrow down the possibilities. All FMRP-SF7 mutant constructs showed an ability to localize to axons, supporting the idea that the region required for axonal localization is in the N-terminus. Intriguingly, the NES34A mutation in which the nuclear export sequence (NES) is deleted showed an increased efficiency over WT to localize axonally. Furthermore, the S500A, S500D and the ΔRGG mutants showed decreased puncta densities in axons. Axonal arbor complexity was not affected in neurons transfected with NES deletion or S500 mutants suggesting these domains may be required for FMRP to function in axon growth and branching regulation. Our results suggest a mechanism of axonal localization of FMRP that requires a region in the N-terminus which allows for all splice forms to be localized and function axonally. A region in the C-terminus is required for FMRP to function in regulation of axonal arbor complexity and this region may be the either the NES or S500 phosphorylation site or both. This is the first known attempt at elucidating a mechanism for differential localization of FMRP splice forms and the role of FMRP domains in axonal localization.M.S., Biology -- Drexel University, 201
