RNA helicase MOV10 is essential for gastrulation and normal CNS development

Skariah, Geena

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https://hdl.handle.net/2142/98256 Copy

Description

Title

RNA helicase MOV10 is essential for gastrulation and normal CNS development

Author(s)

Skariah, Geena

Issue Date

2017-07-09

Director of Research (if dissertation) or Advisor (if thesis)

Ceman, Stephanie

Doctoral Committee Chair(s)

Ceman, Stephanie

Committee Member(s)

• Chung, Hee Jung
• Stubbs, Lisa J.
• Galvez, Roberto

Department of Study

Neuroscience Program

Discipline

Neuroscience

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• Moloney Leukemia Virus 10 (Mov10)
• Ribonucleic acid (RNA) helicase
• Gastrulation
• Brain
• Development

Abstract

Neural circuitry, at its most basic level, is composed of multiple synaptic connections formed between the neurons within a circuit. These synaptic contacts are also the sites of local protein synthesis, which is required for long-term synaptic plasticity underlying learning and memory. The Fragile X Mental Retardation Protein (FMRP) functions as a translational repressor and is critical for fine-tuning local protein synthesis at synapses. Recently, we demonstrated that FMRP associates with the RNA helicase Moloney Leukemia Virus 10 (Mov10) in brain and modulates its translational activity through the microRNA (miRNA) pathway. FMRP is critical for normal cognition and our findings hypothesize a role for Mov10 in brain function. Additionally, Mov10 has a well- documented role of protecting genomic integrity by suppressing actively transposing retroviral elements called LINE-1 (L1) in cell culture systems. This becomes relevant when we consider the studies that propose an increase in endogenous retrotransposition in fetal brain as well as in the hippocampus and other regions of the adult brain during neuronal differentiation. Furthermore, L1 retrotransposition is more common in brain compared to other tissues in the body. Despite the many findings that show a role for Mov10 in suppressing retrotransposition, there are currently no studies describing its role in the developing or adult brain. In order to address theses gaps, we generated a knockout mouse model for Mov10 and found that the deletion of Mov10 causes early embryonic lethality. We were able to show that lethality occurs prior to embryonic day 9.5 establishing a crucial role for Mov10 during mouse embryogenesis. We then used the externally developing Xenopus laevis embryos to establish the exact stage and cause of the Mov10 embryonic lethality. Our findings show that the blocking of translation of the Mov10 maternal mRNAs in one-cell stage embryos results in a gastrulation defect at Stage 10 of Xenopus embryonic development. RNA sequencing of embryos from Stage 10.5 of the Mov10 knockout shows an increase in mRNAs levels consistent with a miRNA-mediated role of Mov10 possibly at the Maternal to Zygotic Transition (MZT). In addition, we see that knockdown of zygotic Mov10 transcripts results in eye and ventricular neuronal differentiation defects in Stage 36 tadpoles. This suggests an important role for Mov10 in postnatal brain development. In agreement with this, we see that WT mice showed a significant increase in Mov10 protein levels in their brains from Postnatal Day 0 (P0) to P14 suggesting an important function for Mov10 during this critical period of synapse formation and neuronal differentiation. Mov10 expression was seen throughout the P1 brain including cortex, hippocampus and cerebellum and became localized mostly to the hippocampus in the adult brain. Interestingly, the subcellular localization of Mov10 was nucleo-cytoplasmic in P1 brains and predominantly cytoplasmic in the adult brain. The same age-dependent change in cellular localization was also observed in cultured neurons from Day In Vitro 0 (DIV0) and DIV14, suggesting a possible nuclear function for Mov10 at postnatal stages. Because of its critical role during development and postnatal increase in the brain, we further examined the Mov10 heterozygotes in this study. We found an increase in L1 genomic DNA content in the Mov10 heterozygote brains at P2 compared to WT brains from the same stage, corroborating Mov10’s role in suppressing L1 retrotransposition. RNA isolation followed by sequencing from Mov10 immunoprecipitates (RNA-IP) at P2 stage in brain shows that Mov10 bound retroelement RNAs belonging to the LINE-1 family. Further verification showed that Mov10 specifically bound a retrotransposition-competent L1 RNA in mouse brain and that it inhibited the cDNA synthesis of this RNA in an in vitro assay. In addition, both the RNA-IP and iCLIP analysis of Mov10 RNA targets at P0-P1 brains followed by functional annotation showed that Mov10 bound significantly more mRNAs involved in cytoskeletal and actin binding. To investigate the role of Mov10 in cytoskeletal dynamics, we created a CRISPR-Cas9 knockout of Mov10 in Neuro2a cells. The deletion of Mov10 in Neuro2a caused abnormally decreased neurite outgrowth on differentiation. We were able to rescue the defect by restoring Mov10 protein levels in the knockout cell line. Furthermore, culturing and staining of hippocampal neurons from Mov10 heterozygotes confirmed these results showing markedly short dendrites as seen in the Mov10 knockout Neuro2a cells. These findings point to an additional stage-specific role for Mov10 in modulating cytoskeletal dynamics that are key to synapse formation and pruning and eventually to the formation of normal brain circuitry. To investigate the behavioral output of reduced Mov10 levels on the brain, we conducted a series of behavioral tests on the Mov10 heterozygotes. We find that the Mov10 heterozygotes show increased activity in a novel environment using the Open-field test as well as increased anxiety in the Elevated- Plus Maze test, suggesting perturbed neuronal circuitry. The Mov10 heterozygotes tested normal in the Trace fear conditioning, Novel Object Recognition and Rotarod tests. These studies along with the neuron culture and knockdown assays suggest that Mov10 is important in developing and maintaining normal brain activity. This is the first study of Mov10 at an organismal level and in the brain.

Graduation Semester

2017-08

Type of Resource

text

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http://hdl.handle.net/2142/98256

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Copyright 2017 Geena Skariah

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