Diversifying Transcription Termination Function

我们的基因被编码在染色体DNA的特定区域。当基因被打开时，它们被复制到一种叫做RNA的相关分子中。基因在DNA双螺旋的特定链上开始和停止。事实证明，被复制的内容并不总是相同的：被复制的序列可以在不同的地方停止（或终止）。这个过程在细胞中被控制，作为调节基因表达的数量和编码的一种方式。例如，最近的研究表明，控制我们生物钟的基因表达节奏是由受调节的终止控制的。尽管终止很重要，但它是复制过程中最不为人所知的方面。出乎意料的是，对植物何时开花的研究为控制终止的方式提供了见解。GGS的实验室最近发现，开花调节因子FPA与一种名为Pcfs2的蛋白质相互作用。Pcfs2与一种名为Pcf11的蛋白质有关，Pcf11在许多生物中都是促进终止的必需蛋白，包括酵母、苍蝇、蠕虫和人类。这一发现的特别之处在于，GGS的实验室发现开花植物进化出了两种相关的Pcf11蛋白（Pcfs2和Pcfs4），而酵母、动物和原始植物似乎只有一种。有趣的是，GGS的实验室发现这两种植物蛋白必须执行特定的任务，因为一种是生命必需的，不与FPA （Pcfs4）相互作用，但另一种不是必需的，但与FPA （Pcfs2）相互作用。这项研究的目的是研究开花植物如何以及为什么进化出两种参与终止的相关蛋白质，并发现它们的工作方式有何不同。这应该为了解植物中基因表达是如何被控制提供基本的见解，并为控制终止的不同方式提供证据，这应该引起广泛的普遍兴趣。本研究的第一个目的是确定Pcfs2和Pcfs4是针对所有基因还是针对不同的基因亚群进行终止。这可以用一种叫做ChIP-seq的方法来完成。然后，我们可以通过对缺乏正常功能的Pcfs2和Pcfs4的突变体中的所有RNA进行测序，来了解这些靶标与功能终止的关系。通过这种方式，我们可以看到哪些基因的复制过程没有正常停止，以及这如何影响邻近基因的表达。我们将整合不同的RNA测序数据来回答这个问题。我们要做的一件事是以前没有人在植物上做过的，那就是在RNA从DNA复制时对其进行排序，这样我们就能立即看到终止过程发生了什么。我们的第二个目标是观察Pcfs4和Pcfs2影响终止的机制是否涉及与不同蛋白质的相互作用，因为它们可能是这一过程中罕见的调节因子。GGS和GJB小组在这一领域组成了一个经验丰富的团队——不仅在理解如何调节终止方面，而且在蛋白质组学和rna测序分析方面取得了突破，这对这项研究至关重要，对其他科学家也很有用。这项工作将为在植物、遗传学、蛋白质组学和大型数据集计算分析方面作为一个团队工作的早期职业科学家提供最先进的培训。这项工作将极大地促进我们对调控终止的新特征的理解，并通过揭示哪些基因受调控终止控制，将开花植物的生物学联系起来。通过这种方式，我们将提供关于基因表达如何在植物中受到控制的基础知识，这对我们未来的粮食和能源安全至关重要。

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.