SMN functions as a molecular chaperone for mRNP assembly Open Access

Donlin-Asp, Paul Gregory (2017)

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Spinal muscular atrophy (SMA) is a neuromuscular disease characterized by a specific degeneration of motor neurons. SMA results from a reduction in the survival of motor neuron (SMN) protein. We have previously shown specific defects in the axonal localization of poly A mRNA (including β-actin and Gap43) and mRNA-binding proteins (HuD, IMP1) in SMN-deficient motor neurons. Our findings led us to hypothesize that SMN plays a role in the assembly of messenger ribonucleoproteins (mRNPs), and that failure to assemble mRNA transport complexes leads to the reported mRNA localization defects in SMA motor neurons.

To test our hypothesis, we have established a trimolecular fluorescence complementation (TriFC) assay as a sensor for the association of mRNAs with RBPs. In motor neurons isolated from a severe SMA mouse model, as well as primary human fibroblasts from SMA patients, we readily detect a defect in the assembly of complexes containing IMP1 protein and β-actin mRNA. Furthermore, RNA immunoprecipitation experiments also show impairments in the association of IMP1 protein with β-actin mRNA. Through biochemical fractionation, we observe a consistent shift of IMP1-containing mRNPs toward smaller granules in SMA human fibroblasts. In SMA patient derived fibroblasts, IMP1 granules are consistently reduced in their volume relative to control lines, a phenotype consistent with both our TriFC and fractionation results. Finally, we can show a defect in the association of IMP1 with the cytoskeleton in the SMA patient fibroblasts, suggesting a mechanism to explain reduced mRNA localization reported in SMA motor neurons.

In summary, our results show that SMN plays a more general role in RNP assembly beyond the canonical role in snRNP assembly. Here, we demonstrate that SMN acts as a chaperone for the formation of transport-competent RNA granules, providing a mechanism for mRNA localization defects that may contribute to the motor neuron degeneration observed in SMA.

Table of Contents

Table of Contents--

1. A role for the survival of motor neuron protein in mRNP assembly and transport 1

Abstract 2

Introduction 3

Spinal Muscular Atrophy is characterized by axonal and synaptic defects 5

The SMA disease protein SMN has an essential role in splicesomal snRNP assembly 6

SMN in actively transported in axons during development 8

SMN is required for the axonal delivery of mRNPs 9

SMN is involved in translational regulation 12

Does SMN regulate the assembly of mRNP granules? 13

Conclusion 14

Figures 16

Figure 1. Mechanisms of axonal mRNA localization and local translation 16

Figure 2. Model for the mislocalization of mRNA observed in SMA motor neurons 18

Figure 3. SMN in mRNP assembly and axonal mRNA localization 20

2. Spatially and temporally regulating translation via mRNA binding proteins in cellular and neuronal function 22

Abstract 23

Introduction 24

1. Polarity- from the basal to apical membranes and beyond. 25

2. Translational regulation in neuronal function 28

2.1 Dendrites 28

2.2 Axons 29

3. Dysregulation of local translation in disease 30

3.1 Neurodevelopmental diseases 30

3.2 Neurodegenerative diseases 32

3.3 Axonal injury and repair 35

4. Studying translation regulation: the ever expanding toolbox 35

4.1 Determining where mRNA is in space and time 36

4.2 Defining what are mRNA binding proteins and what mRNAs they bind 38

4.3 Defining what transcripts are being translated and where translation occurs 40

Conclusions and future directions 43

Figures 44

Figure 1. Translation Repression in RNA localization 44

Figure 2. Local translation in cellular polarity 46

Figure 3. Local translation in neuronal function 48

Figure 4. Dysregulation of local translation in disease 50

3. The survival of motor neuron protein acts as a molecular chaperone for mRNP assembly 52

Summary 53

Highlights 55

Background 56

Results 57

Association of IMP1 protein with β-actin mRNA is impaired in cultured motor neurons from a SMA mouse model 57

IMP1 mRNP granules show assembly defects and reduced size in SMA patient fibroblasts 60

IMP1 containing mRNP granules are reduced in volume in SMA patient fibroblasts 62

IMP1 mRNP granules show decreased association with the cytoskeleton in SMA patient fibroblasts 62

Discussion 64

Experimental Procedures 69

Acknowledgments 75

Figures & Tables 77

Figure 1. Trimolecular Fluorescence Complementation (TriFC) allows visualization of RNA and protein association in situ 78

Figure 2. IMP1 and β-actin association is reduced in an SMA mouse model 81

Figure 3. IMP1 and β-actin association is reduced in SMA patient fibroblasts 83

Figure 4. IMP1 association with mRNA is impaired in SMA patient fibroblasts 85

Figure 5. IMP1 granules show reduced complexity in SMA patient samples 88

Figure 6. IMP1 granules are reduced in size in SMA patient samples and can be rescued by restoring expression of SMN 90

Figure 7. IMP1 complexes show reduced association with the cytoskeleton in SMA fibroblasts 93

Figure 8. SMN functions as a chaperone for IMP1-β-actin granule assembly 97

Supplementary Figure 1. Cytoskeletal proteins show unaltered distributions in SMA fibroblasts 99

Supplementary Figure 3. PABPC1 granules show reduced complexity in SMA patient samples 101

Supplementary Table 1. Statistical comparisons for figure 1D 103

Supplementary Table 2. Statistical comparisons for figure 1E 105

Supplementary Table 3. Statistical comparisons for figure 3B 107

Supplementary Table 4. Statistical comparisons for figure 4B, IMP1 steady state protein levels 110

Supplementary Table 5. Statistical comparisons for figure 4B, SMN steady state protein levels 113

Supplementary Table 6. Statistical comparisons for figure 4E 116

Supplementary Table 7. Statistical comparisons for figure 6D 123

Supplementary Table 8. Statistical comparisons for figure 6F 126

Supplementary Table 9. Statistical comparisons for figure 7B 131

Supplementary Table 10. Statistical comparisons for figure 7D 134

Supplementary Table 11. Statistical comparisons for figure 7E 137

Supplementary Table 12. Statistical comparisons for figure 7G 139

Supplementary Table 13. Statistical comparisons for figure 7I 141

4. Conclusions and future directions 148.

Overview 149

SMN as a chaperone for RNP assembly 150

What mRNA binding proteins are altered in their association with mRNA in SMA? 152

What RNP complexes show alterations in their assembly in SMA? 153

What extent in vivo does non-splicing related RNP changes contribute to pathology in SMA? 154

Are defects seen in SMA mouse motor neurons are conserved in patient derived samples? 156

Future directions for SMA therapy 157

Why are motor neurons affected in SMA? 160

Materials and methods 161

Figures 164

Figure 1. Widespread RNA dysfunction likely contributes to SMA pathology and motor neuron death 164

Figure 2. SMN in RNP biogenesis 166

Figure 2. RNA interactome capture to assess mRNP granule composition in SMA patient samples 168

Figure 3. RNP gradient isolation to assess widespread changes in RNP granule size and complexity in SMA patient samples 170

Figure 4. SMA IPSC motor neuron cultures show defects seen in SMA mouse motor neurons 172

Figure 5. Manipulating mRNA levels as a therapeutic strategy in SMA 174

Figure 6. RG3039 increases TriFC signal in cell culture 176

Figure 7. RG3039 increases poly(A) RNA signal in treated cells 178

Figure 8. Hypo vs Hyper RNP assembly in disease 180

5. Appendix. 182

mRNAs are mislocalized from axonal compartments with SMN deficiency 183

GAP43 protein is reduced in SMA growth cones 183

Overexpression of IMP1 and HuD rescues GAP43 axonal deficiency 184

SMN deficiency affects translation at the growth cone 185

Materials and methods 187

Figures 192

Figure 1. Gap43 and β-actin mRNAs are reduced in axons and growth cones of SMN deficient motor neurons. 192

Figure 2. GAP43 protein levels are reduced in axonal growth cones of SMA motor neurons 194

Figure 3. SMN deficiency causes reduced local protein synthesis in axonal growth cones 196

Figure 4. Overexpression of HuD and IMP1 restores GAP43 protein and transcript levels in growth cones, as well as axon outgrowth of SMA motor neurons 198

6. References 200

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