Localization, Mechanisms and Functions of SMN and hnRNP-Q1 Open Access

Xing, Lei (2010)

Permanent URL: https://etd.library.emory.edu/concern/etds/t722h958n?locale=en
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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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