Award details

Functional RNAs in Axis Formation

Reference	BB/L007525/1
Principal Investigator / Supervisor	Professor Karuna Sampath
Co-Investigators / Co-Supervisors
Institution	University of Warwick
Department	Warwick Medical School
Funding type	Research
Value (£)	688,519
Status	Completed
Type	Research Grant
Start date	05/05/2014
End date	04/11/2017
Duration	42 months

Abstract

Functional RNAs, which include protein coding and non-coding RNAs, are beginning to be recognized to play key roles in many biological processes. How non-coding RNAs and other functional RNAs regulate early development is poorly understood. Patterning of metazoan embryos requires both maternal and zygotic factors. Since gene expression is minimal during early embryogenesis in many animals, maternal factors play key roles. These maternal RNA or protein factors elicit their functions before the zygotic genome is activated. We recently identified a non-coding activity in the 3' un-translated region (3'UTR) of maternal RNA encoding the secreted Nodal morphogen, Squint (Sqt), that regulates formation of embryonic dorsal structures in zebrafish. Maternal sqt is localized to presumptive dorsal cells. The 3'UTR of sqt RNA can induce dorsal gene expression in early embryos, independent of the signaling functions of Sqt/Nodal protein. Dorsal non-coding activity of sqt does not require Sqt protein or Nodal signaling, but requires maternal Wnt/beta-catenin signaling. This activity is mediated through a sequence motif in the sqt 3'UTR. Our findings suggested that maternal sqt acts as a functional RNA with roles in dorsal axis formation. The mechanisms by which sqt RNA elicits its biological effects are unknown. Therefore, the proposed project will specifically address the following questions: How does non-coding maternal sqt RNA function in specification of dorsal structures? How is the non-coding activity of maternal sqt regulated? How does maternal sqt interact with the Wnt/beta-catenin pathway? Which proteins bind to sqt RNA to specify embryonic dorsal? Coding and non-coding RNAs in the genome play important roles in many organisms including humans. Thus, the proposed project on the non-coding function of a coding RNA can be a paradigm for understanding how RNAs function independently of their protein-coding role.

Summary

The proposed project aims to understand the basis of a very fundamental and crucial process, i.e., how does the egg of an animal know where to place the head and spine structures, and how does it go about it. Ribonucleic acids (RNA) are an essential component of all cells. We are working on an RNA whose location predicts where the head and back structures will form in developing zebrafish. Misplacing the RNA or blocking the functions of the RNA results in fish embryos lacking these essential parts. Therefore, this RNA has a very crucial role in formation of the head and spine structures in this organism. We now want to understand precisely how the RNA controls these events. Similar RNAs exist in humans as well. We use the zebrafish as a model organism to understand this process because we can obtain embryos are externally fertilized and develop outside the body of the mother, which allows us to watch these processes as they take place, both under normal conditions and upon perturbation by various means. We will make various changes in the RNA and observe how these changes affect formation of head and spine structures. Specifically, we will either remove certain segments in the RNA or alter them by scrambling the segments, to learn if these segments are crucial to the function of the RNA in making the head and spinal cord. Since early fish embryos are optically clear, we can observe the RNA as it moves to its correct location in live samples in a non-invasive manner by taking videos. Zebrafish can lay hundreds of eggs at a time, a feature that allows us to easily extract the RNA and anything that binds to it. By knowing which factors are associated with the RNA, we can get insights into how it may function. Since similar RNAs exist in humans, it is possible that dysfunctions of such RNAs might result in birth defects. Therefore, the proposed project addresses a very fundamental question, and has implications for human congenital birth defects.

Impact Summary

Impact Summary: Our research will have a broad range of impacts, from advancement of fundamental scientific knowledge, with implications for human birth defects, to training of highly skilled workforce. Contribution to economic competitiveness and quality of life: The proposed project aims to understand the basis of a fundamental process, and has potential for novel discoveries. The project will make important contributions to scientific knowledge and understanding of non-coding RNA functions, with potential implications for human congenital birth defects arising from dysfunctions in non-coding/functional RNAs. Training of skilled workforce: A key output of this project will be training of staff. The PDRA and RA will be trained in many advanced skills in molecular genetics, RNA-protein biochemistry, developmental biology, imaging and proteomic data analysis. This is a highly specialized and valuable skill-set, and will equip the PDRA for a career in academia or industry. The University of Warwick provides a wide range of transferable skills courses that are designed to maximize individual potential and employability. Thus, we will contribute to the training of a highly skilled workforce. Communications and Public engagement: My laboratory in Singapore trained ~50 secondary school and undergraduate students over a year period, and will continue to do so at the BCB, WMS. The WMS has an active public engagement policy and a good track record in outreach activities. We will present our work to the public through exhibitions, open days, school visits, by setting up a project website and making podcasts suitable for lay members of the public. We will work with the WMS Marketing and Communications office in order to ensure that our material is accessible and suitable. Public lectures on the importance and implications of our science will be given through Café Scientifique (Leamington Spa), the local forum of a national network aimed at debating science issues and promoting public engagement with science. Collaborations and Co-Production: Data generated from the project will provide sufficient opportunities for future collaborations. The approaches described and data will be of interest to biochemists, researchers working on RNA biology and non-coding RNAs, and developmental biologists. We will establish new collaborations with relevant interested groups as opportunities arise. Resource Generation: This project will generate many new resources of interest to other researchers: peptides, plasmid constructs, antibodies etc. These will be made available, after publication, to the broader community. We will also generate mutant and transgenic lines, which will be distributed via the Zebrafish Resource Centres. Data from mutant analysis will be shared through publications, and made available via web-based portals such as the ZFIN, EUFishBiomedNet, and the Zebrafish Phenome Project. Some of the peptide data may be of potential commercial value or of interest to industry. Exploitation and Application: If any findings of potential commercial interest are identified, we will work with Warwick Ventures, the Technology Transfer Office of the University of Warwick, in order to protect IPR before dissemination, and to explore routes to exploitation via commercial partnerships and pathways. Impact activity deliverables & milestones: Work described in this project will contribute towards scientific knowledge, and will increase our understanding of non-coding RNA functions in normal development, with potential implications for human birth defects. We will publish the findings arising from this work in high quality manuscripts, in Open Access journals where possible, and make our results available to the broader scientific community via seminars, poster/platform presentations at scientific conferences. The work will generate a variety of reagents and information. All outputs will be made available on request, after publication.

Committee	Research Committee C (Genes, development and STEM approaches to biology)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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