Abstract
dc:description.abstractRibonucleic acid (RNA) is a biological molecule that exists in the cell of virtually all kinds of known living organisms. Although its significance in fundamental cellular processes such as protein synthesis has been known for decades, an increasing number of novel species and functions of RNA have been discovered recently, and a complete picture of RNA functions in the cell is still elusive. One of the major challenges in RNA studies is that there had been no efficient method to quantify RNA with spatial and temporal information in vivo. In my doctoral studies, I developed a novel RNA sequencing method that metabolically labels RNA in a cell- and time-specific manner in mice and applied this method to solve biological problems that had been difficult to tackle. First, the robustness of the newly developed in vivo RNA labelling method was assessed. To confirm the sensitivity and specificity of RNA labelling, multiple transgenic mice that label RNA in different cell types were generated. By comparing the data obtained from each transgenic strain to previously generated transcriptomic datasets, I confirmed that RNA labelling in a specific cell type was achieved in all the strains analysed. This method would be useful to study cell-type-specific transcriptomics rather than the commonly used laborious and time-intensive cell isolation method often used, and might provide data that closely reflect the native transcriptional state in vivo. Next, using the same RNA labelling method, I tested if there is any RNA that is mobile between different cell types in mice. Intercellular mobility of RNA has been shown in nematodes and plants, but whether there is any RNA that is mobile between different mammalian cells in vivo is still unclear. The cell-specific RNA labelling method allowed us to assess the mobility of RNA directly for the first time. Based on previous publications, three different cell types were chosen as potential “donor” cells, and transgenic mice that label RNA in these cells were generated. The donor cell-derived labelled RNA was sought in “recipient” tissues that are not capable of labelling RNA. However, although RNA labelling was achieved in the donor tissues, no labelled RNA was found in the recipient tissues in any of the animals tested. This experiment presents a novel methodology to assess the mobility of RNA in living mice, and the obtained data suggest that only minor intercellular transfer of RNA, at best, is happening in the tested pairs of tissues. In the final part of my thesis, I applied the metabolic RNA labelling method to study the transcriptional dynamics in the early preimplantation mouse embryos. Unlike conventional RNA sequencing methods that can only quantify RNA abundance in each stage of the embryos, metabolic RNA sequencing can directly interrogate the transcriptional activity of each gene. This method is particularly powerful in studying the transcriptional network in the early preimplantation embryo, where embryo-derived RNA needs to be distinguished from maternally-deposited RNA. By exposing the mouse embryos to a nucleotide analogue in a stage-specific manner, I identified genes that are actively transcribed in the 2-cell embryos. This method would be useful in studying the transcriptional cascade in the early mammalian preimplantation embryos.
Degree
thesis:*- Name dc:type.qualificationname
- Doctor of Philosophy (PhD)
- Level dc:type.qualificationlevel
- Doctoral
- Grantor dc:publisher.institution
- University of Cambridge
- Year dc:date.issued
- 2020
Author and committee
dc:creator, dc:contributor.*- Author dc:creator
-
- Matsushima, Wayo
- Advisor dc:contributor.advisor
-
- Miska, Eric
Subjects
dc:subject × 8Rights
dc:rightsIdentifiers
dc:identifier.*- Author Identifier
- 0000-0002-0334-2423
- OAI identifier oai:identifier
- oai:www.repository.cam.ac.uk:1810/306780