The RNA-binding protein, ZC3H14, is critical for control of polyadenylation and translation, neuronal development, brain morphology, and working memory in mice Público
Rha, Jennifer (2017)
Abstract
A number of mutations in genes that encode ubiquitously expressed RNA-binding proteins cause tissue specific disease, many of which are neurological, suggesting critical roles for this class of proteins in the brain. We recently identified mutations in a gene that encodes a ubiquitously expressed polyadenosine RNA-binding protein, ZC3H14 (Zinc finger CysCysCysHis domain-containing protein 14), that cause a nonsyndromic, autosomal recessive form of intellectual disability. This finding reveals the molecular basis for disease and provides evidence that ZC3H14 is essential for proper brain function. To investigate the role of ZC3H14 in the mammalian brain, we have generated a Zc3h14 knockout mouse. We show here that Zc3h14 is not essential in mice. Utilizing these mice, we provide the first in vivo functional characterization of ZC3H14, as a regulator of RNA poly(A) tail length. The Zc3h14D/D mice show defects in brain structure as well as working memory. Proteomic analysis comparing the hippocampi of Zc3h14+/+ and Zc3h14D/D mice reveal dysregulation of several pathways that are important for proper brain function and shed light onto which pathways are most affected by the loss of ZC3H14. This newly generated mouse provides a model to study the function of ZC3H14 in the brain and thus elucidating how mutation in Zc3h14 could lead to intellectual disability.
Table of Contents
Chapter 1: Introduction to Dissertation..........................................................................1
1.1 Molecular basis of learning and memory................................................................2
1.2 RNA regulation in the brain....................................................................................4
1.3 ZC3H14.……………………………………..........................................................7
1.3.1 Historical Background: From budding yeast to humans.................................7
1.3.2 Phylogeny and Structure: Evolutionarily conserved functional domains........8
1.3.3 Molecular Function: ZC3H14 regulates poly(A) tail length..........................10
1.3.4 ZC3H14 in Disease: Mutations in ZC3H14 cause autosomal recessive intellectual disability…………….………………………………………………..12
1.4 Summary and prevailing questions…………………………................................13
Chapter 2: The RNA-binding protein, ZC3H14, is required for proper polyadenylation, expression of synaptic proteins, and brain function in mice………24
2.1 Introduction............................................................................................................25
2.2 Results....................................................................................................................27
2.2.1 Generation and confirmation of Zc3h14D/D mice.............................................27
2.2.2 ZC3H14 is not essential but is required for normal litter and testis size.........30
2.2.3 Zc3h14 is required for proper poly(A) tail length control..............................31
2.2.4 Zc3h14D/D mice show structural defects in the brain.......................................31
2.2.5 Zc3h14D/D mice have impaired working memory but intact learning..............32
2.2.6 Zc3h14D/D mice have normal visual function and exhibit normal motor function and coordination..............................................................................................33
2.2.7 Zc3h14D/D mice exhibit increased resistance to seizures.................................35
2.2.8 Increased expression of synaptic proteins in Zc3h14D/D mice………………..36
2.3 Discussion..............................................................................................................38
Chapter 3: The evolutionarily conserved RNA-binding protein dNab2 interacts with the Fragile X protein homolog and mediates translational repression in Drosophila neurons…………………………………………………………………………………..66
3.1 Introduction............................................................................................................67
3.2 Results....................................................................................................................71
3.2.1 dfmr1 is a dominant modifier of dNab2 overexpression in the eye................71
3.2.2 dfmr1 interacts with dNab2 in locomotor behavior and mushroom body development.....................................................................................................72
3.2.3 dNab2 co-localizes with dFMRP RNPs in neurites.........................................74
3.2.4 The dNab2 and dFMRP proteins physically associate in neurons……….…..76
3.2.5 dFMRP and dNab2 co-regulate target RNAs..................................................77
3.2.6 ZC3H14 localizes to axons and associates with the translational machinery..79
3.3 Discussion..............................................................................................................81
Chapter 4: ZC3H14 in neuronal development and synapse formation.....................107
4.1 Introduction..........................................................................................................108
4.2 Results..................................................................................................................109
4.2.1 Young ZC3H14-deficient hippocampal neurons develop fewer and shorter neurites in culture...........................................................................................109
4.2.2 ZC3H14 localizes to dendritic spines in mature cultured hippocampal neurons...........................................................................................................109
4.2.3 Loss of ZC3H14 causes abnormal spine morphology and density………....109
4.2.4 Loss of ZC3H14 causes increase in number of dendritic shaft synapses…..110
4.3 Discussion............................................................................................................110
Chapter 5: Discussion: Brief summary, what we've learned, more questions, and future directions.............................................................................................................122
5.1 Learning about learning and memory...................................................................123
5.2 Discussion of answers to questions posed at the beginning of our investigation...124
5.3 Concluding remarks.............................................................................................134
Chapter 6: Material and Methods................................................................................142
6.1 Chapter 2..............................................................................................................143
6.2 Chapter 3..............................................................................................................157
6.3 Chapter 4..............................................................................................................163
Chapter 7: References...................................................................................................164
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