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Developmental Analysis of DEAD Box 1 Open Access

Descriptions

Other title
Subject/Keyword
DEAD box
pre-implantation development
RNA granules
Ribonuclease
Type of item
Thesis
Degree grantor
University of Alberta
Author or creator
Hildebrandt, Matthew R
Supervisor and department
Godbout, Roseline (Oncology)
Examining committee member and department
Godbout, Roseline (Oncology)
Hazes, Bart (Medical Microbiology and Immunology)
McDermid, Heather (Biological Sciences)
Owttrim, George (Biological Sciences)
Rancourt, Derrick (Oncology and Biochemistry & Molecular Biology)
Department
Department of Oncology
Specialization
Experimental Oncology
Date accepted
2014-09-22T10:12:30Z
Graduation date
2014-11
Degree
Doctor of Philosophy
Degree level
Doctoral
Abstract
DEAD box protein 1 (DDX1) is a member of the DEAD box family of helicases which bind and unwind double-strand (ds) RNA and are associated with all aspects of RNA metabolism. DDX1 was identified by differential screening of a subtracted cDNA library prepared from two retinoblastoma (RB) cell lines. DDX1 is co-amplified with MYCN and overexpressed in a subset of MYCN-amplified neuroblastoma (NB) cell lines and tumours as well as in all MYCN-amplified RB cell lines examined to date. DDX1 has been shown to be associated with a number of different cellular processes including DNA repair and mRNA 3’ processing, polyadenylation and transport. In vitro work has demonstrated a role for DDX1 in unwinding RNA-RNA and RNA-DNA duplexes (up to 29 base pairs) in an ADP-dependent manner. Finally, DDX1 can digest single-strand (ss) RNA in a magnesium-dependent and energy-independent manner. The structures of a number of DEAD box proteins have been solved. These X-ray structures have been used to determine the mechanisms by which DEAD box proteins bind and modify RNA and ribonucleoprotein (RNP) complexes. I was able to generate a number of DDX1 protein crystals including some obtained in the presence of an RNA-DNA duplex; however, none of these crystals yielded diffraction patterns. An in-depth characterization of the ribonuclease activity of DDX1 revealed that DDX1 degrades single-strand (ss) RNA in a sequence-independent manner. My results suggest that DDX1 is a ribonuclease that cleaves ssRNA down to the 3-6 nucleotide range. Attempts to map the ribonuclease activity of DDX1 identified the carboxyl-terminal region of DDX1 as being essential for ribonuclease activity. I used a gene knockout model to examine the role of DDX1 during development. I found that Ddx1 knockout embryos stall during development at the 2- or 4-cell stage. Intriguingly, I also noted that a subset of the wild-type embryos die between E3.5 and E6.5. Wild-type embryos that were dying arose from second generation intercrossed heterozygote mice. This phenomenon was observed in both the FVB and C57BL/6 mouse strains and represents a type of non-Mendelian inheritance not reported previously. Examination of the subcellular localization of DDX1 in fetal and adult mouse tissues revealed a primarily nuclear localization. However, when I examined the subcellular localization of DDX1 in oocytes and pre-implantation embryos, I found that the protein was localized primarily to the cytoplasm where it formed large aggregates. These aggregates were highly dynamic in nature, with size and numbers changing as a function of developmental stage. While I was not able to identify any organelles or proteins that co-localized with DDX1 in the aggregates, I was able to determine that the aggregates at the 2-cell stage were dependent on the presence of RNA. DDX1 aggregates in the cytoplasm of pre-implantation embryos may therefore represent a novel RNA containing granule that plays a role either in the protection or degradation of RNAs.
Language
English
DOI
doi:10.7939/R3C24QW19
Rights
This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for the purpose of private, scholarly or scientific research. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.
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