Browsing by Subject "mRNA degradation"
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Item mTOR dependent regulation of Kv1.1 in normal and disease states by the RNA binding factors, HuD and miR-129-5p(2014-05) Sosanya, Natasha Marie; Raab-Graham, Kimberly F.; Atkinson, Nigel; Golding, Nace; Haris, Adron; Iyer, VishwanathLittle is known about how a neuron undergoes site-specific changes in intrinsic excitability in normal and diseased conditions. We provide evidence for a novel mechanism for the mammalian Target of Rapamycin Complex 1 (mTORC1) kinase dependent translational regulation of the voltage-gated potassium channel Kv1.1 mRNA (Chapter 2). First, we identified a microRNA, miR-129-5p, that represses Kv1.1 mRNA translation when mTORC1 is active. When mTORC1 is inactive, we found that the RNA-binding protein, HuD, binds to Kv1.1 mRNA and promotes its translation. Surprisingly, mTORC1 activity does not alter levels of miR-129 and HuD to favor binding to Kv1.1 mRNA but affects the degradation of high-affinity HuD target mRNAs, freeing HuD to bind Kv1.1 mRNA. Thus, high affinity HuD target mRNAs can serve two purposes under normal physiological conditions: 1) to provide functional proteins, such as CaMKIIα, that change the architecture of the synapse and 2) serve as a sponge sequestering HuD from translating mRNAs like Kv1.1. To determine if this mechanism for repression of Kv1.1 expression is conserved in a disease model where mTORC1 activity is overactive, we assessed the expression levels of active mTORC1, Kv1.1, and miR-129-5p in a rat model of temporal lobe epilepsy (TLE; Chapter 3). We found that when mTOR activity is low in TLE, Kv1.1 expression is high and behavioral seizure number is low. In contrast, when behavioral seizure activity starts to rise there is a corresponding increase in mTOR activity and Kv1.1 protein levels dramatically drop. In addition, we found that miR-129-5p, the negative regulator of Kv1.1 mRNA translation increases by 21 days post status epilepticus (SE) to sustain Kv1.1 mRNA translational repression. Thus, long-term changes in Kv1.1 protein levels result in a hyperpolarized threshold for action potential firing. Our results suggest that increased mTOR activity following SE results in two phases of Kv1.1 repression (1) in an initial repression of Kv1.1 mRNA translation by mTOR activity that is followed by (2) an onset of elevated miR-129-5p expression that sustains Kv1.1 repression. These studies suggest that dynamic changes in miR- 129-5p provide potential novel targets for epilepsy interventions. mTOR is a protein kinase that promotes CaMKIIα mRNA translation (Sosanya et al., 2013; Chapter 2); however, the mechanism and site of dendritic expression are unknown. Herein (Chapter 4), we show that mTOR activity mediates the dendritic branch specific expression of CaMKIIα, favoring one secondary, daughter branch over the other in a single neuron. Notably, reduction in mTOR activity decreases the overall dendritic expression of CaMKIIα protein and RNA through the shortening of its poly(A) tail. Overexpression of HuD both increases total CaMKIIα levels and rescues the selective expression of CaMKIIα in one daughter branch over the other. These results suggest that differential branch targeting of HuD may mediate the branch specific expression of CaMKIIα in neuronal dendrites during mTOR activity. Furthermore, when mTOR activity is reduced HuD releases CaMKIIα mRNA and thus exposes its poly(A) tail to be deadenylated, reducing its overall expression and eliminating its branch specific expression.