Role of 3' untranslated regions in translation regulation of GluR2 mRNAs 公开

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

About this Dissertation

Rights statement

• Permission granted by the author to include this thesis or dissertation in this repository. All rights reserved by the author. Please contact the author for information regarding the reproduction and use of this thesis or dissertation.

School	Laney Graduate School
Department	Graduate Institute of the Liberal Arts
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Biology, Neuroscience Health Sciences, Pharmacology
关键词	mRNA seizure 3'UTR pilocarpine GluR2
Committee Chair / Thesis Advisor	Dingledine, Raymond J, Emory University
Committee Members	Hall, Randy A, Emory University Feng, Yue, Emory University Murphy, T J, Emory University

最新修改

Primary PDF

Thumbnail	Title	Date Uploaded	Actions
	Role of 3' untranslated regions in translation regulation of GluR2 mRNAs ()	2018-08-28 12:51:26 -0400	Press to Select an action Download

Supplemental Files

Thumbnail	Title	Date Uploaded	Actions