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Role of DDX1 in Embryonic Development
- Author / Creator
- Wang, Yixiong
DEAD Box 1 (DDX1) is a member of the DEAD box protein family of RNA helicases involved in all aspects of RNA metabolism. Although several DEAD box proteins have been well characterized, the biochemical activity of the majority of DEAD box proteins remains to be studied. DDX1 is mostly localized in the nucleus of most cell types analysed; however, it is also abundant in the cytoplasm of tumours in which it is overexpressed. DDX1 plays a variety of roles ranging from RNA transport to the repair of DNA double stranded breaks. Ddx1 knockout results in abnormal development with reduced body size and aberrant gametogenesis in Drosophila, and embryonic lethality in mouse.
My PhD research project is focused on the characterization of DDX1 in early developmental stages of mouse embryos. In Chapter 2, we report that DDX1 forms large cytoplasmic aggregates that do not co-localize with any known organelle or cytoplasmic granule markers in embryos, with the exception of stress granule markers TIA-1 and TIAR. We also show that Ddx1-/- mouse embryos stall at the 2- to 4-cell stages in vitro. Given that DDX1 is an RNA binding protein, and maternal RNAs are essential for early embryonic development, we pursued a possible relationship between DDX1 aggregates and RNAs. Using RNase A treatment, we obtained evidence that the role of DDX1 in mouse embryos is RNA dependent. By carrying out DDX1-RNA immunoprecipitation and qPCR analysis, we found that DDX1 binds to a subset of maternal RNAs that are critical for early-stage embryonic development.
In Chapter 3, we further characterized the role of DDX1 in early-stage mouse embryos using electron microscopy (EM) and Stimulated Emission Depletion (STED) microscopy. The latter provides greatly enhanced resolution compared to conventional confocal microscopy. With STED microscopy, we showed that DDX1 forms aggregates at the subplasmalemmal cytoplasm of 2-cell embryos and forms ring-like clusters. With EM, we discovered that DDX1 localizes to previously uncharacterized membrane bound vesicles. These vesicles contain an electron dense RNA core as well as Ca2+. We named these vesicles MARVs (Membrane Associated RNA-containing Vesicles). In Ddx1-/- embryos, we observe greatly reduced numbers of MARVs and misregulation of Ca2+, which results in the upregulation of reactive oxygen species (ROS) in mitochondria, as well as increased nuclear and mitochondrial fragmentation. Given that DDX1 binds to maternal RNAs in 2-cell embryos, and Ca2+ indirectly regulates the phosphorylation of cytoplasmic polyadenylation factor CPEB1, we examined whether cytoplasmic polyadenylation factors were present in MARVs. Intriguingly, we found that two cytoplasmic polyadenylation factors, CPEB1 and CPSF2, co-localize with DDX1. Because CPEB1 and CPSF2 are critical for the initiation of the cytoplasmic polyadenylation process, our results suggest that MARVs may be hubs for cytoplasmic polyadenylated RNAs, with DDX1 either protecting these RNAs until they are required or playing a role in polyadenylation.
In Chapter 4, we address the importance of DDX1 in early embryonic development. We show that, in contrast to our in vitro work, Ddx1-/- embryos do not survive past the 1-cell stage in vivo. To further characterize DDX1-binding RNAs, we carried out DDX1 RNA-immunoprecipitation using lysates prepared from 2-cell embryos, followed by HiSeq analysis. Surprisingly, approximately 20% of the transcripts identified by RIP-seq were processed pseudogenes and ~24% were long non-coding RNAs. As pseudogenes and long non-coding RNAs are important in the generation of endogenous siRNAs in early embryonic development, we propose that DDX1 may function with these pseudogenes in the protection or processing of RNAs.
These studies reveal critical roles for DDX1 and MARVs in early mouse embryonic development. Future work will focus on the mechanism of action of DDX1 and MARVs vis-a-vis Ca2+ regulation, cytoplasmic RNA polyadenylation and endogenous siRNAs, all of which are essential for early embryonic development. Given that in yeast and human cells DEAD box proteins also regulate RNA-containing phase-separated structures, which are now known to be membrane-less organelles, future work will address whether the RNA cores that reside in MARVs are phase-separated RNA droplets that control the fate of maternal RNAs during early embryonic development.
- Graduation date
- Fall 2021
- Type of Item
- Doctor of Philosophy
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