%0 Journal Article
%T Identifying intrinsic and extrinsic determinants that regulate internal initiation of translation mediated by the FMR1 5' leader
%A Tara Dobson
%A Erika Kube
%A Stephanie Timmerman
%A Les A Krushel
%J BMC Molecular Biology
%D 2008
%I BioMed Central
%R 10.1186/1471-2199-9-89
%X Cellular transfection of a dicistronic DNA construct containing the FMR1 5' leader inserted into the intercistronic region yielded significant translation of the second cistron, but the FMR1 5' leader was also found to contain a cryptic promoter possibly confounding interpretation of these results. However, transfection of dicistronic and monocistronic RNA ex vivo or in vitro confirmed that the FMR1 5' leader contains an IRES. Moreover, inhibiting cap-dependent translation ex vivo did not affect the expression level of endogenous FMRP indicating a role for IRES-dependent translation of FMR1 mRNA. Analysis of the FMR1 5' leader revealed that the CGG repeats and the 5' end of the leader were vital for internal initiation. Functionally, exposure to potassium chloride or intracellular acidification and addition of polyinosinic:polycytidylic acid as mimics of neural activity and double stranded RNA, respectively, differentially affected FMR1 IRES activity.Our results indicate that multiple stimuli influence IRES-dependent translation of the FMR1 mRNA and suggest a functional role for the CGG nucleotide repeats.The mRNA and protein generated from the FMR1 gene in neurons is localized to dendrites [1,2]. The FMR1 protein, FMRP is synthesized in response to neural activity and its function as an RNA binding protein influences the translational level of other dendritically localized mRNAs [3-6]. FMRP is also part of the RISC complex [7,8], a set of proteins that interact with micro-RNAs or short interfering RNAs to inhibit translation and or degrade the RNA, respectively.Regulating the synthesis of FMRP is important for cellular function. FMRP over-expression leads to a defect in dendritic architecture, synaptic differentiation, and abnormal behaviors in mice and flies [9,10]. Alternatively, the absence of FMRP in Fragile X Syndrome (FXS) leads to alterations in synaptic plasticity resulting in mental retardation [11]. FXS develops from an expansion of the CGG nucleotide rep
%U http://www.biomedcentral.com/1471-2199/9/89