Fragile X syndrome (FXS) is the most common monogenic cause of intellectual disability. FXS is marked by hyperactivity and approximately 25% of patients have epilepsy, but the underlying molecular causes are unknown. FXS is caused by transcriptional silencing of the fragile X mental retardation gene 1 (FMR1), resulting in the loss of the fragile X mental retardation protein (FMRP). FMRP is an mRNA binding protein involved in the regulation of mRNA translation. Recent findings show that FMRP regulates the expression of the A-type potassium channel Kv4.2, which plays an essential role to regulate hippocampal excitability, suggesting that dysregulated expression of Kv4 channels may cause epilepsy and hyperexcitability in patients with FXS. To date, it is unclear whether FMRP acts as a translational activator or inhibitor of Kv4.2 mRNA. We hypothesize that mRNA translation and levels of the potassium channel Kv4.2 are dysregulated in a human cell model of FXS. Here, we have analyzed (1) Kv4.2 expression levels and (2) Kv4.2 mRNA translation in a human in vitro cell model for FXS. We tested how the manipulation of FMRP levels with short interfering RNA (siRNA) sequences, or by overexpression of FMRP in a human cell line affects Kv4.2 expression and mRNA translation. Our initial analyses of human Kv4.2 protein expression in vitro using a luciferase reporter system showed decreased expression of Kv4.2 protein after knockdown of FMR1. However, polysome association studies suggested that Kv4.2 mRNA translation was unchanged after knockdown of FMR1. In contrast, FMRP overexpression led to a decrease in Kv4.2 expression and mRNA translation in both assays. Based on these and previous studies, we propose a new model for the regulation of Kv4.2 mRNA by FMRP via two separate binding sites in its 3'UTR. We hypothesize that FMRP acts as a translational activator at the proximal binding site, and a translational inhibitor at the distal site. Preliminary experiments support this hypothesis. This study provides evidence that FMRP is an important regulator of Kv4.2 expression in human cells, and has also led to the development of a novel, testable model of how FMRP regulates the mRNA translation of the specific target mRNA Kv4.2.
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About this Honors Thesis
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|Effect of FMRP deficiency on the expression and mRNA translation of the potassium channel Kv4.2 in human cells ()||2018-08-28||