Role of 3' untranslated regions in translation regulation of GluR2 mRNAs Open Access
Irier, Hasan A (2009)
Abstract
GluR2 expression is regulated at the transcription level by
cell-specific
transcription factors that target the promoter, and GluR2 is also
subject to
translational control by the GU repeats residing in the long 5'
untranslated region
(UTR). In this study, the translational regulation of GluR2 mRNAs
by alternative
3'UTRs was explored. GluR2 mRNAs exist as two major GluR2
transcripts of 6
kb and 4 kb, differing only in the length of their 3'UTRs (~2750 bp
or "long" and
~750 bp or "short", respectively) in rats and mice. Both short and
long GluR2
mRNAs are abundantly expressed in CA1 and CA3 pyramidal neurons,
and
dentate granule cells (DG). Pilocarpine-induced status epilepticus
(SE)
significantly reduced GluR2 mRNA levels in CA1 and CA3 but not DG.
In
Xenopus oocytes, the expression profiles of luciferase
reporters bearing
alternative GluR2 5' and 3' UTRs were studied. In the absence of
long 5'UTR,
which contains translation repressor elements, the long 3'UTR
serves as a
translational suppressor for GluR2 transcripts. In rat hippocampus
, the majority of
endogenous GluR2 transcripts exhibited strong association with
polysomes,
which is indicative of active translation, whereas GluR2
transcripts bearing long
3'UTRs were associated with ribosome-free ribonucleoprotein
complexes. A de-
repression of translation of GluR2 mRNAs bearing long 3'UTRs after
prolonged
seizures was observed. The mechanism of the long 3'UTR mediated
translation
repression was studied using the luciferase reporter mRNAs bearing
alternative
GluR2 UTRs in rabbit reticulocyte lysates treated with translation
elongation
inhibitors and translation initiation modulators. Translation of
the reporter mRNAs
bearing the long GluR2 3'UTR was insensitive to low concentrations
of the
elongation inhibitors cycloheximide and anisomycin, in contrast to
a reporter
bearing the short 3'UTR, which was inhibited, suggesting that
initiation is the site
of translation regulation for GluR2 mRNAs bearing the long 3'UTRs.
The
translation initiation modulator kasugamycin selectively induced
the expression of
reporter mRNAs bearing either of the long UTRs of GluR2. These
findings overall
suggest that GluR2 transcripts have distinct translation patterns
due to
alternative 5' and 3'UTRs. The mechanisms of UTR-mediated
translation
regulation present potential targets for therapeutic modulation of
GluR2
expression in a transcript-specific manner.
Table of Contents
TABLE OF CONTENTS
Chapter I: Background and significance …………………………………………..............1
A. Glutamate receptors: Classification & Biological Functions……..............1
a. Ionotropic Glutamate Receptors……………………………...........2
b. Ionotropic glutamate receptors in health and disease….................6
c. Identification and Physiological Function of AMPA receptors……8
d. Significance of GluR2 subunit in functional AMPA receptors….....11
B. Role of untranslated regions of mRNA in RNA processing…….................13
a. 5' UTRs…………………………………………..………….…..........17
b. 3'UTRs ………………………………………………..…….…..........22
C. Molecular Control of GluR2 subunit expression…………………................26
a. Transcriptional Regulation of GluR2 gene……………….................26
b. Translational Regulation of GluR2 mRNA…………………..............30
D. Goals of my thesis research……………………………………...….............31
Chapter II: Materials and Methods………………………………………………..................32
A. Materials……………………………………………………………….............32
B. Methods………………………………………………………………..............33
a. Preparation of GluR2 3'UTR constructs……………………..............33
b. Reporter expression and RNA stability in Xenopus oocytes……....34
c. In vitro translation and RNA stability of reporters in rabbit
reticulocyte lysates (RRL)………………………………….………....35
d. Sucrose gradient analysis of endogenous GluR2 transcripts……...37
e.Quantitative Real Time PCR analysis of reporter and native
GluR2 mRNAs………………………………………………....……...38
f. Subcellular distribution of endogenous GluR2 transcripts………….39
g. Northern blot analysis of native GluR2 transcripts……..………........40
h. Non-isotopic in situ hybridization of native GluR2 mRNAs…………40
i. Pilocarpine-induced status epilepticus……………….……..…….....41
Chapter III: Translational regulation of GluR2 mRNAs in rat hippocampus
by alternative 3' untranslated regions ………………………………………….42
A. Abstract………………………………………………………………..……......42
B. Introduction………………………………………………………….....……......43
C. Results…………………………………………………………….………….....45
a. GluR2 mRNA bearing long 3'UTR is the majority GluR2 species
in rat hippocampus but is underrepresented in the cytoplasm……...45
b. Translation profile in rat hippocampus of native GluR2 transcripts
bearing alternative 3'UTRs……………………....…………....……….51
c. Translational regulation by alternative GluR2 3'UTRs……...................54
d. Effect of pilocarpine-induced status epilepticus (SE) on translation and
regional distribution of GluR2 transcripts with alternative 3'UTRs……61
D. Discussion…………………………………………………………….………......83
Chapter IV: Control of GluR2 translational initiation by its alternative 3'UTRs……........…….86
A. Abstract……………………………………………………………….......……....86
B. Introduction……………………………………………………………......……....87
C. Results…………………………………………………………………......……...89
a. Effects of elongation inhibitors on translation of GluR2
reporter mRNAs………………………………………………....……......89
b. Effects of initiation modulators on translation of GluR2 reporters……105
D. Discussion……………………………………………………………....……......110
a. Effects of DMDA-Pateamine A on the GluR2 mRNA
translation initiation……………………………………………………...113
b. Transcript-specific alternative initiation of GluR2 mRNA
translation by Kasugamycin……………………………......……….......113
Chapter V: Summary and Future Directions………………………………….......………........115
A. Summary of major findings.…………………………………………..….……….......115
B. Future Directions…………………………………….…………………………….......118
a. Role of 3'UTR-interacting proteins on expression profile of
luciferase reporter mRNAs bearing alternative GluR2 UTRs
in Xenopus oocytes..………………….……………………………..........118
b. Determining the GluR2 mRNAs bearing predominant combinations
of 5'-and 3'UTRs in rat hippocampus……...……………..........................135
c. What is the mechanism underlying seizure-induced translational
de-repression of GluR2 mRNAs that are translationally restricted
due to presence of long 5' or long 3'UTRs? ……………………………..146
C. Implications……………………………………………….……………….…………….146
Chapter VI: References …………………………………………………………….……………..148
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