Award details

Diversifying Transcription Termination Function

Reference	BB/M004155/1
Principal Investigator / Supervisor	Professor Gordon Simpson
Co-Investigators / Co-Supervisors	Professor Geoffrey Barton
Institution	University of Dundee
Department	School of Life Sciences
Funding type	Research
Value (£)	778,082
Status	Completed
Type	Research Grant
Start date	01/06/2015
End date	31/05/2019
Duration	48 months

Abstract

Termination is the least understood aspect of the transcription cycle, but is a newly emerging level at which eukaryotic gene expression is regulated. An unexpected by-product of the genetic analysis of flowering time control has been the identification of a series of factors that enable flowering, and control cleavage, polyadenylation and transcription termination. The analysis of such factors has yielded insight into the regulation of RNA 3' end formation that is of wide general interest. We recently discovered that the flowering regulator FPA directly interacts with Pcfs2, a protein related to the conserved termination factor Pcf11. In contrast to other eukaryotes that encode a single, essential Pcf11 protein, flowering plants have evolved two proteins related to Pcf11 that are diversified in function: Pcfs2 interacts with FPA and is non-essential, while Pcfs4 does not interact with FPA and is essential. In this proposal we aim to understand how and why the function of a crucial termination factor has diversified in flowering plants. We will identify the immediate targets of Pcfs2 and Pcfs4 using ChIP-Seq and validate the functional impact of these associations through integration of specialised RNA-sequencing datasets (including nascent transcriptomes) designed to interrogate defective termination. We will combine this approach with a cross-link based proteomics procedure and in vitro binding assays to determine which regulatory proteins Pcfs2 and Pcfs4 associate with. This research will advance our fundamental understanding of the mechanisms controlling transcription termination and how it can be regulated. The integration of multiple, specialized RNA-Seq reactions and sequencing of the nascent transcriptome will influence the design and interpretation of future transcriptome studies. In the longer term, this study will link back to biology, revealing targets and process controlled by gene-specific termination.

Summary

Our genes are encoded in specific regions of DNA in our chromosomes. When genes are switched on, they are copied into a related molecule called RNA. Genes start and stop at specific stretches of a particular strand of the DNA double helix. It turns out that what is copied is not always the same: the sequence being copied can stop (or terminate) at different places. This process is controlled in the cell as a way to tune how much gene expression occurs and what will be coded. For example, it was very recently shown that the rhythms of gene expression that run our body clock are controlled by regulated termination. Despite its importance, termination is the least understood aspect of the copying process. Unexpectedly, the study of when plants flower has provided insight into ways in which termination can be controlled. GGS's lab recently discovered that the flowering regulator FPA interacts with a protein called Pcfs2. Pcfs2 is related to a protein called Pcf11 that is known to be essential for promoting termination in many organisms, including yeast, flies, worms and humans. What is special about this finding is that GGS's lab discovered flowering plants have evolved two related Pcf11 proteins (Pcfs2, Pcfs4), while yeast, animals and primitive plants appear only to have one. Intriguingly, GGS's lab discovered that these two plant proteins must carry out specialized tasks because one is essential to life and doesn't interact with FPA (Pcfs4), but the other is not essential, but does interact with FPA (Pcfs2). The aim of the research proposed here, is to work out how and why flowering plants have evolved two related proteins involved in termination and to discover how they work differently. This should provide basic insight into how gene expression is controlled in plants and provide evidence of different ways in which termination can be controlled that should be of wide general interest. The first objective of this study is to determine whether Pcfs2 and Pcfs4 target all genes or different sub-sets of genes for termination. This can be done using a method called ChIP-seq. We can then tell how these targets relate to a function in termination by sequencing all the RNA in mutants that lack properly functioning Pcfs2 and Pcfs4. In this way we can see at which genes the copying process does not stop properly and how this affects the expression of neighbouring genes. We will integrate different RNA sequencing data to answer this question. One thing we will do that no one has ever done in plants before is sequence the RNA as it is being copied from DNA, so we can immediately see what is happening to termination. Our second objective is to see if the mechanism by which Pcfs4 and Pcfs2 affect termination involves interaction with different proteins because these could be rare examples of regulators of this process. The GGS and GJB groups form a hugely experienced team in this area - not only in understanding how termination can be regulated, but also in developing breakthroughs in proteomics and RNA-sequencing analysis essential to this study and which are generally useful to other scientists. This work will provide state-of the-art training for early career scientists working as a team on plants, genetics, proteomics and computational analysis of large datasets. This work will greatly advance our understanding of novel features of regulated termination and link back to the biology of flowering plants by revealing what genes are controlled by regulated termination. In this way, we will provide underpinning knowledge about how gene expression is controlled in plants essential to our future food and energy security.

Impact Summary

1. Cultural Life. Our work defines a new area of science aiming to uncover why flowering plants have evolved two related termination factors diversified in function. This curiosity-led discovery of new knowledge is a feature that the UK public expect of their scientists as GGS experienced when he spoke about non-coding antisense RNAs at a BBSRC organized public engagement event at the Edinburgh International Science Festival. 2.Agricultural Industry. Our work benefits the development of world agriculture in several distinct ways. First GGS's lab is training a new generation of plant scientists familiar with working with genetics, making crosses and phenotyping plants. Second, we are training biologists used to working in multi-disciplinary teams, combining the iterative interaction of bench scientists with computational biologists. Third, through analysis of huge sequencing datasets we are drawing into plant biology, scientists from mathematical and physics backgrounds, who bring with them quite different skill-sets and insight that can be highly beneficial to understanding plant biology and hence crop science. Fourth, we are developing cross-link based in vivo interaction proteomics. In light of the relative challenges in extracting proteins from living plants compared to human cell lines, this development may be particularly beneficial to plant biologists. Finally, GGS's position is jointly supported by The James Hutton Institute. Dedicated crop science colleagues are immediately exposed to the work of his lab in Arabidopsis and this has facilitated translation to biofuel research, barley genetics, potato disease and the timing of raspberry fruit development. 3. World Economy. Dundee takes the training of PhD and Post-Doctoral scientists particularly seriously and has a specific department called "OPD" that delivers "non-bench" training in, for example, public speaking and public engagement. Dundee provides a highly international working environment with staff from over 60 different nationalities working in the College of Life Sciences. Dundee also houses the 3rd largest biotech cluster in the UK with an entrepreneurial culture of spin-out companies from Life Sciences. Together, these aspects of research life in Dundee provide rounded, highly skilled and educated employees to the international work-force. For example, among recent alumni from GGS's lab who came to Dundee from overseas, C. Hornyik has gone on to hold a P.I. position in crop science in the UK and L. Terzi a managerial position in a Swiss pharmaceutical company. 4. Society Through Public Engagement. This proposal relates to fundamental understanding of plant gene expression. The GM controversy highlights the importance of public understanding and support for the research we do. GGS became responsible for Dundee Plant Sciences impact activities in 2010 and since then the Division has successfully developed valuable links with Dundee's Botanic Garden, a hands-on DNA extraction activity to communicate information about plants having genes and a sustainable "Genetics Garden". This has involved all members of GGS's lab and most members of the Division of Plant Sciences. Further links with the Botanic Garden are planned through annual Family Fun Days, Fascination for Plants Days and the annual replanting of variants of our Genetics Garden. In this proposal we specifically describe a computer generated animation of the processes of cleavage, polyadenylation and termination of RNA. Using this, we will also communicate very specifically our research interest in gene expression to the general public. The work of our groups in public engagement is not unique, but part of the culture of Dundee University College of Life Sciences, reflected by the fact that Dundee won the inaugural BBSRC "Excellence with Impact" competition and are participants in the current competition.

Committee	Research Committee B (Plants, microbes, food & sustainability)
Research Topics	Plant Science
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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