Regulation of A-type potassium channel Kv4.2 expression by FMRP and miR-324-5p Público
Yao, Xiaodi (2012)
Published
Abstract
Regulation of A-type potassium channel Kv4.2 expression
Fragile X syndrome (FXS) is the most common form of inherited
intellectual disability
that is caused by loss of the Fragile X mental retardation protein
(FMRP), a RNA binding
protein involved in various aspects of mRNA regulation. One
prominent characteristic of
FXS is high susceptibility to seizure resulting from neuronal
hyperexcitability; yet the
underlying molecular mechanisms remain elusive. Potassium channels
play critical roles
in controlling neuronal excitability. Recent findings suggest that
FMRP might directly
regulate voltage-gated potassium channels. Voltage-gated Kv4.2
potassium channel is the
major component mediating the A-type K+ outward current thereby
controlling neuronal
excitability in the hippocampus and has been implicated in
epilepsy. Thus we
investigated the possible involvement of FMRP to regulate Kv4.2
that might explain the
high susceptibility to epilepsy in FXS. In this dissertation we
uncovered two novel
posttranscriptional regulation mechanisms to modulate the
expression of Kv4.2. We show
that FMRP associates with Kv4.2 mRNA and positively regulates Kv4.2
mRNA
translation and protein expression in neurons. Moreover, both total
and cell surface
protein levels of Kv4.2 are reduced in a mouse model of FXS. Our
results suggest that
loss of FMRP results in Kv4.2 downregulation which might contribute
to excess neuronal
excitability in Fmr1 KO mice and thus imply a potential
mechanism underlying FXS-
associated epilepsy. In contrast to FMRP, the microRNA (miRNA)
miR-324-5p was
shown to be an inhibitory regulator for Kv4.2 expression in
neurons. Our results show
that miR-324-5p targets to the 3'UTR of Kv4.2 mRNA and inhibits
Kv4.2 protein
expression. Furthermore, miRNA-324-5p
overexpression reduced surface expression of Kv4.2 in neurons.
Taken together, our
results suggest that miR-324-5p may regulate Kv4.2 expression and
functions during
neuronal development and plasticity. Collectively, my thesis work
provides new insights
into the molecular mechanisms in regulating neuronal excitability
and eplepitogenesis.
Dysregulation of Kv4.2 expression through two mechanisms may be
involved in epilepsy.
Our findings therefore provide a molecular basis for future studies
toward a better
understanding the interplay of these mechanisms in the pathogenesis
of FXS and other
forms of epilepsy.
Table of Contents
Table of Contents
Chapter I. General
Introduction………………………………………………………..1
Section 1.1 Fragile X syndrome and FMR1 gene
…………………….………..…2
Section 1.2 The FMRP
Protein……………….......……………………………….4
Section 1.3 FMRP is an RNA binding
protein………………………….………...5
Section 1.4 Functions of FMRP in mRNA metabolism and
dynamics……...…..8
Section 1.4.1 FMRP represses
translation………………………………9
Section 1.4.2 Possible mechanism of FMRP-mediated
translational
inhibition………………………..………………………………………..13
Section 1.4.3 FMRP positively regulates
translation……………….…...16
Section 1.4.4 FMRP regulates mRNA
stability……………….…….…...17
Section 1.4.5 FMRP regulates mRNA
transport…..………………….….18
Section 1.4.6 FMRP is involved in mRNA
editing…………...…………21
Section 1.4.7 FMRP functions in protein-protein
interaction…………....21
Section 1.5 Roles for FMRP in neuronal and synaptic
function………..…….…23
Section 1.6 FMRP and
epilepsy…………………………….……………….…..27
Section 1.7 Kv4.2 potassium channel, a possible link between FMRP
and
epilepsy…………...…………….………………………………………………..28
Section 1.7.1 The role of Kv4.2 for synaptic
function…………….…….29
Section 1.7.2 The role of Kv4.2 in
epilepsy…………………….……....30
Section 1.8 Thesis
objectives………………………………………………...….32
Section 1.9 Figures and
legends…………………………………………………34
Chapter II. Fragile X Mental Retardation Protein Regulates
Protein Expression and
mRNA Translation of the Potassium Channel
Kv4.2...………………………………38
Section 2.1
Abstract………………………………………………………….…..39
Section 2.2
Introduction……………………..……………………………….…..40
Section 2.3 Materials and
Methods……………………………………………....42
Section 2.4
Results…………………………………………………..……….…..45
Section 2.5
Discussion……………………………………………….......………49
Section 2.6
Acknowledges……………………………..…………………….…..52
Section 2.7 Figures and
legends……………………………………………..…..53
Chapter III. miR-324-5p regulates the expression of the
potassium channel
Kv4.2……………………………………………………………………………………62
Section 3.1
Introduction…………………………………………………….…..63
Section 3.2 Materials and
Methods……………………………………………..67
Section 3.3
Results………………………………………………………….…..70
Section 3.4
Discussion…………………………………………….…...………..75
Section 3.5 Figures and
legends………………………………………...……….79
Chapter IV. Summary and Future
Directions………………………………………..87
Chapter V.
References……………………………………………………..…….…….97
Table of Figures
Figure 1-1 A schematic view of the FMR1
gene……………………………………...…34
Figure 1-2 A schematic of FMRP protein
…….……………………………………...….36
Figure 2-1 Kv4.2 protein levels are reduced in Fmr1 KO
hippocampal dendrites ……...53
Figure 2-2 Reduced levels of Kv4.2 at neuronal cell surfaces in
Fmr1 KO mice can be
partially rescued by antagonizing mGlu5.
……………………………………..……..…55
Figure 2-3 Kv4.2 mRNA translation is reduced in synaptic fractions
from Fmr1 KO
cortices.………………………………………………………………………………..…57
Figure 2-4 FMRP regulation of Kv4.2 mRNA is mediated by both
5′- and 3′-UTRs of
Kv4.2 mRNA
…………………………………………………………………………....59
Figure 3-1 Kv4.2 mRNA is a putative target of miR-324-5p.
………………………….79
Figure 3-2 Point mutation in the seed region of Kv4.2 mRNA
abolishes the function of
miR-324-5p Kv4.2 mRNA.
………………………………………………..……………81
Figure 3-3 miR-324-5p is expressed in the hippocampus
………...………………….....83
Figure 3-4 miR-324-5p regulates the total and cell surface levels
of Kv4.2. …………...85
Figure 4-1 Proposed models for the cooperation of FMRP and
miR-324-5p in the
regulation of Kv4.2
expression.….………………………………………………….…...95
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