Localization, Mechanisms and Functions of SMN and hnRNP-Q1 Público
Xing, Lei (2010)
Published
Abstract
Diverse types of messenger ribonucleoprotein (mRNP) complexes/granules, exist in the cytoplasm to enable the post-transcriptional regulation of gene expression e.g. mRNA transport granules, stress granule and P-bodies. However, their inter-relationships are unclear. Various mRNA binding proteins have been shown to influence mRNP granule assembly and/or regulate the recruitment and shuttling of target mRNAs. The cellular functions of mRNA binding proteins localized to mRNP granules and mechanisms underlying mRNA regulation are poorly understood. Some components of mRNP granules are affected in genetic diseases, such as the Survival of Motor Neuron protein (SMN), reduced levels of which leads to a motor neuron degenerative disease, Spinal Muscular Atrophy (SMA). The best understood SMN function is to facilitate spliceosome assembly. The overall objectives of my thesis were to characterize components of cytoplasmic RNA granules, assess their dynamic inter-relationships, and elucidate novel cellular functions of component mRNA binding proteins. This thesis research led to the discovery of an SMN-Gemin multiprotein complex within axonal transport granules, which are deficient of spliceosomal proteins. The mRNA binding protein, hnRNP-Q1, which directly interacts with SMN, was shown to be a novel component of SMN transport granules in neuronal processes, further suggesting a non-canonical function of SMN in axonal mRNA regulation. The results further showed that hnRNP-Q1 is a component of multiple RNA granules, and associates with several compartmented mRNAs. We revealed an unexpected role of hnRNP-Q1 to regulate RhoA signaling and found that after hnRNP-Q1 knockdown, hippocampal neurons exhibited a reduced dendritic spine density, and C2C12 cells exhibited enhanced cell spreading and increased focal adhesions and stress fibers. These phenotypes mimic effects observed upon the activation of the RhoA/ROCK signaling cascade and were rescued by a ROCK antagonist. Further studies demonstrated that hnRNP-Q1 associates with RhoA mRNA and negatively regulates its translation. hnRNP-Q1 depletion upregulated both RhoA protein levels and activities of the RhoA/ROCK signaling pathway. Collectively, this dissertation research has uncovered novel functions of mRNA granule components in mRNA regulation and should further motivate studies directed to understand the complex cytoplasmic regulation of mRNA underlying cellular development and diseases.
Table of Contents
Chapter I. General
Introduction………………………………………………………..1
Section 1.1 mRNPs and related
granules…………………………………………..…2
Section 1.1.1 Transport mRNP
granules………………………………………….3
Section 1.1.2 Stress
granules……………………………………………………...6
Section 1.1.3 Processing
bodies…………………………………………………..8
Section 1.2 mRNA localization in polarized cells provides mechanism
for protein
sorting……………………………………………………………………………………10
Section 1.3 Molecular mechanisms of mRNA localization: interactions
between
cis-acting elements and mRNA binding
proteins………………………………………..13
Section 1.4 RNA-binding proteins in mechanisms of translational
regulation……...16
Section 1.5 RNA-binding proteins regulating mRNA
degradation………..…….…..20
Section 1.6 RNA-binding proteins and
diseases……………….………………….…24
Section 1.6.1 Spinal Muscular Atrophy (SMA) and Survival of Motor
Neuron
protein……………………………………………………………………………………25
Section 1.7 Outline of this
dissertation……………………………………....…...….31
Chapter II. Multiprotein Complexes of the Survival of Motor
Neuron Protein
SMN with Gemins Traffic to Neuronal Processes and Growth Cones
of
Motor
Neurons………………………………………………………………………….36
Section 2.1
Abstract……………………………………………………………….…37
Section 2.2
Introduction………………………………………………………….…..38
Section 2.3 Materials and
Methods……………………………………………….….40
Section 2.4
Results……………………………………………………………….…..48
Section 2.5
Discussion……………………………………………………………….57
Section 2.6
Acknowledge…………………………………………………………....62
Section 2.7 Figures and
Legends………………………………………………….…63
Chapter III. hnRNP-Q1 regulates cellular morphogenesis by
inhibiting RhoA mRNA
translation………………………………………………………………………81
Section 3.1
Introduction………………………………………………………….…..82
Section 3.2 Materials and
Methods…………………………………………………..83
Section 3.3
Results……………………………………………………………….…..92
Section 3.4
Discussion……………………………………………………...………100
Section 3.5 Figures and
Legends……………………………………………….…..109
Section 3.6 Supplemental
Figures……………………………………………….….123
Chapter IV. Microscopic analysis and biochemical
characterization of hnRNP-Q1-containing mRNP
granules………………………………………………………...…128
Section 4.1
Introduction…………………………………………………………….129
Section 4.2 Materials and
Methods………………………………………………....132
Section 4.3
Results……………………………………………………………….…136
Section 4.4
Discussion……………………………………………………………...145
Section 4.5 Figures and
Legends…………………………………………………...154
Section 4.6 Supplemental
Figures…………………………………………………..163
Chapter V. Summary and Future
Directions………………………………………..170
Section 5.1
Summary……………………………………………………………….171
Section 5.2 Future
Directions………………………………………………………176
Chapter VI.
References……………………………………………………………….186
Table of Figures
Figure 1-1 Proposed dynamic interaction of mRNA-containing granules
and
polyribosomes……………………………………………………………………………32
Figure 1-2 Loss of human SMN1 leads to
SMA……………………………………...….34
Figure 2-1 Colocalization of endogenous SMN and Gemin proteins in
neurites and growth cones in primary forebrain culture and ES-cell
derived motor neurons……..…..63
Figure 2-2 Colocalization of endogenous SMN and Gemin2 in axons and
dendrites of differentiated cultures of primary hippocampal and
motor
neurons…………………..…65
Figure 2-3 Quantitative analysis of SMN-Gemin colocalization in
growth cones following 3D
reconstruction…………………………………………………………..…66
Figure 2-4 SMN granules within neurites are deficient of Sm
proteins
in motor
neurons………………………………………………………………………....68
Figure 2-5 Colocalization and co-transport of fluorescently tagged
and overexpressed SMN and
Gemin2……………………………………………………………………..…69
Figure 2-6 FRET analysis depicts interaction between EYFP-SMN
and
ECFP-Gemin2 or ECFP-Gemin3 in
neuritis……………………………..........................71
Figure 2-7 Gemin2 is recruited by SMN into granules within
neurites and growth
cones………………………………………………………………..73
Figure 2-8 Interactions between Gemin2 with SMN necessary for
recruitment into granules and stabilize
Gemin2………………………………………………………...…75
Figure 2-9 Association of SMN and hnRNP-Q1 in 293 cells and
hippocampal
neurons………………………………………………………………….....78
Figure 2-10 Co-transport and colocalization of mRFP-SMN and
EGFP-hnRNP-Q1
in live rat hippocampal
neurons………………………………………………………....80
Figure 3-1 Western blot analysis of axonal fractions purified from
cultured cortical neurons and synaptic fractions from
brain……………………………………………..109
Figure 3-2 Localization of hnRNP-Q1 granules in neuronal
processes
and growth
cones…………………………………………………………..…………..110
Figure 3-3 Selective enrichment of mRNAs in immunoprecipitated
Flag-mCherry-hnRNP-Q1 containing
complexes……………………………………………………...112
Figure 3-4 hnRNP-Q1 knockdown leads to reduced spine density of
mature mouse hippocampal
neurons……………………………………………………………...……113
Figure 3-5 Knockdown of hnRNP-Q1 in C2C12 cells by RNAi leads to
enhanced cell spreading and reduced
motility………………………………………………………....115
Figure 3-6 hnRNP-Q1 knockdown leads to enhanced spreading of newly
plated C2C12
cells……………………………………………………………………………………..116
Figure 3-7 Effects of ROCK inhibitor Y-27632 on the morphology of
C2C12 cells transfected with control and hnRNP-Q1
siRNA……………………………………….117
Figure 3-8 hnRNP-Q1 knockdown leads to enhanced focal adhesion
and
stress fiber
formation……………………………………………………………….…..118
Figure 3-9 hnRNP-Q1 knockdown upregulates RhoA protein expression
and
Cofilin
phosphorylation…………………………………………………………...……119
Figure 3-10 Knockdown of hnRNP-Q1 leads to exaggerated RhoA
mRNA
degradation……………………………………………………………………………..120
Figure 3-11 Distribution of RhoA and γ-actin mRNAs in linear
sucrose gradients……122
Supplemental Figures 3-1 Western blot analysis of overexpressed
EGFP-tagged
hnRNP-R, hnRNP-Q3, hnRNP-Q1 and hnRNP-Q1 with C-terminus
deletion with
hnRNP-Q1…………………………………………………………………………..….123
Supplemental Figures 3-2 Efficient knockdown of hnRNP-Q1 in mature
mousehippocampal neurons by RNAi.
……………………………………………..….124
Supplemental Figures 3-3 Knockdown of hnRNP-Q1 in C2C12 cells has
no obvious effect on
microtubules…………………………………………………………….….....125
Supplemental Figures 3-4 Enhanced cell spreading and upregulated
RhoA
expression in primary mouse embryonic fibroblasts with hnRNP-Q1
knockdown…....126
Supplemental Figures 3-5 hnRNP-Q1 knockdown did not affect
overall
polysome
profiles…………………………….……………….…………………….…..127
Figure 4-1 Subcellular distribution of hnRNP-Q1, hnRNP-Q3 and
hnRNP-R….….….154
Figure 4-2 Association of hnRNP-Q1 with mRNP
components……………….………155
Figure 4-3 Active transport of EGFP-hnRNP-Q1 in neuronal
processes…….………...156
Figure 4-4 Localization of hnRNP-Q1 and SMN to arsenite-induced
stress granules....158 Figure 4-5 Localization of hnRNP-Q1 to
P-bodies…………………….………………159
Figure 4-6 Differential localization of RNA binding proteins to
P-bodies…….……....160
Figure 4-7 Co-immonuprecipitation analysis of indicated RNA binding
proteins
with Dcp1a and
LSm1………………………………………………………………….162
Supplemental Figure 4-1 Domain structures and sequences of
hnRNP-Qs
and
hnRNP-R……………………………………………………………………….…..163
Supplemental Figure 4-2 Efficient elimination of endogenous RNAs by
S7
nuclease
treatment………………………………………………………………………165
Supplemental Figure 4-3 Western blot analysis of immuprocipitated
Myc-tagged
proteins for mRNA
quantification……………………………...………………………165
Supplemental Figure 4-4 Relative enrichment of mRNAs normalized to
Myc-Q1(1-161) immuprecipitation
pellet………………………………………………………………..166
Supplemental Figure 4-5 Knockdown of hnRNP-Q1 did not affect the
formation of arsenite-induced stress
granules……………………..………………………………….167
Supplemental Figure 4-6 Knockdown of hnRNP-Q1 did not affect
P-body
formation……………………………………………………………………….168
Supplemental Figure 4-7 Co-immonuprecipitation of EGFP-tagged
hnRNP-Q1, Zbp1, HuD, FMRP, PABPC1 and Pumilio2 (Pum2) with
Dcp1a…………………………….169
Figure 5-1 The role of hnRNP-Q1 in regulating the RhoA/ROCK
signaling pathway...184
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