Expression of fragile X mental retardation‐1 gene with nuclear export signal mutation changes the expression profiling of mouse cerebella immortal neuronal cell

Abstract

Fragile X syndrome (FXS) is the most frequent cause of inherited mental retardation and is largely caused by a loss of expression of fragile X mental retardation protein (FMRP), encoded by fragile X retardation gene-1 (Fmr1). FMRP is a multifunction protein, with intrinsic RNA-binding properties, which is a component of ribonucleoprotein complex associated with polyribosomes. The properties of FMRP indicate that it might participate in post-transcriptional processes in the regulation of some mRNA species, including localization, stability and translational control. However, the function of FMRP related to the pathologenesis in FXS is largely unknown. Many efforts were undertaken to identify the putative specific RNA targets as well as the FMRP-related proteins and to identify the effect of FMRP absence on the corresponding proteins. Here we present our efforts using proteomics approach to explore the differential expression profiling of mouse cerebella immortal cell, in which we changed the expression of FMRP by expressing Fmr1 gene with nuclear export signal (NES) mutation. This mutation makes FMRP unable to shuttle from nucleus to cytoplasm and leads to nuclear instead of cytoplasmic location as usual, which was hypothesized to affect the pathways of groups of RNAs or proteins related with FMRP. In present study, 56 proteins were found to be differentially expressed in transfected R2 neuronal cells, including 16 decreased expressions and 40 increased expressions. The differentially expressed proteins play roles in diverse physiological processes, such as neuronal plasticity, spermatogenesis and craniofacial and limb development etc. In addition, the expressions of three mRNA identified as FMRP targets in fragile X cell were tested in present model cells. All these results provide new insights to the role of FMRP in the disease.

Ultrastructural analysis of the functional domains in FMRP using primary hippocampal mouse neurons

“…Several experiments have shown that FMRP without a NES (FMRP-NES) accumulates in the nucleus in different cell lines (Fridell et al, 1996; Hu et al, 2005; Tamanini et al, 1999a; Willemsen et al, 1996), although some studies also have shown the presence of small quantities of FMRP-NES in the cytoplasm (Kim et al, 2009). Fmr1 KO neurons transfected with EGFP-FMR1_Iso12 showed a predominantly nuclear localization, while the dendrites did not show dendritic EGFP-FMRP_Iso12-positive RNA-granules.…”

The Potential for Proteomics in Understanding Neurodevelopmental Disorders

“…Proteomic studies in FXS animal models have indeed provided evidence for alterations of several proteins associated with the central nervous system [53][54][55][56][57]. In a FXS mouse cerebellum cell the expression of the fragile X mental retardation protein (FMRP) was altered, creating a model for cellular deficits found in FXS.…”

“…In a FXS mouse cerebellum cell the expression of the fragile X mental retardation protein (FMRP) was altered, creating a model for cellular deficits found in FXS. The investigators found 56 proteins differentially expressed in the transfected cells, including 16 proteins whose levels were decreased and 40 whose levels were increased [53]. Using SILAC, a study of primary neuronal culture taken from an FXS knock-out mouse demonstrated changes in more than 100 proteins, many of which were associated with nervous system function [58].…”

Investigação de elementos regulatórios do éxon 14 do gene Fmr1 e análise de expressão de suas isoformas

“…3-B A superexpressão da FMRP recombinante sem o éxon 14 leva a sua retenção no núcleo celular (Eberhart et al, 1996;Willemsen et al, 1996;Tamanini et al, 1999;Davidovic et al, 2006), possivelmente devido à ausência do NES localizado neste éxon (Sittler et al, 1996). Mutagênese dirigida ao NES confirmou a necessidade deste para a localização citoplasmática da FMRP (Fridell et al, 1996;Bardoni et al, 1997;Hu et al, 2005;Ramos et al, 2006). Tanto a deleção apenas do éxon 14, quanto a mutagênese dirigida ao NES não avaliam a mudança de ORF causada pela exclusão do éxon 14 por splicing altertivo.…”