Tuning gene expression through antisense transcript dynamics

基因表达是如何控制的？这是分子生物学中最基本的问题，几十年来一直被深入研究。压倒性的范式一直是，表达受到转录因子的调控，转录因子与调控DNA结合，打开或关闭相关基因。大约10年前，人们意识到这样的过程可能会受到噪音的显著影响，这一认识加强了这一框架，但并未颠覆。然而，最近大规模测序基因组时代的实验已经开始揭示这一范式中的空白。特别是，这些实验揭示了大多数基因组都是普遍转录的，因此不仅蛋白质编码基因，而且基因组的许多其他区域都被转录，产生所谓的非编码RNA。因此，DNA通常不仅在制造蛋白质所需的方向上转录(正义方向)，而且还在相反的方向(反义方向)转录。然后问题就出现了，所有这些额外的转录都在做什么：它是一个意外，还是嘈杂的蜂窝环境不可避免的副产品，或者它起到了某种调节功能？越来越多的人接受这一结论，即它确实具有重要的监管作用。然而，非编码的、通常是反义的RNA所起的实际作用还不清楚。以前的大多数研究都是在整个基因组的水平上探索这些问题，因此很难得出关于特定基因调控的结论。在这个项目中，我们建议以植物开花基因FLC为背景，密切关注反义RNA的调控。FLC是一种开花抑制基因，其定量转录水平对于确保植物在繁殖成功的最佳时间开花至关重要。在正常情况下，准确的转录水平被认为是由两个反义非编码RNA控制的。在这里，我们试图了解这两个反义RNA的差异生产如何能够调节FLC基因的表达。我们试图证明(或反驳)的一种可能性是，每个单独的FLC基因在很长一段时间内只产生两个反义转录之一，并随着时间的推移在一种状态和另一种状态之间随机来回切换。这两种不同的状态被认为对FLC基因的表达状态有非常不同的影响。通过控制系统处于一种或另一种(双稳)状态的时间长度，然后可以精确地调节正义FLC的表达水平。我们将检验这一假说(和其他假说)，以前所未有的细节揭示非编码RNA如何作为表达的数量调节因子发挥作用。如果我们能深入到这一机制的核心，我们就更接近于回答我们开始的问题：基因表达是如何控制的？

How is gene expression controlled? This most fundamental of questions in molecular biology has been intensively studied over many decades. The overwhelming paradigm has been that expression is regulated by transcription factors that bind to regulatory DNA to switch on or off associated genes. The realisation about 10 years ago that such processes can be significantly influenced by noise has augmented but not overturned this framework. However, recent experiments from the genomic era of massive sequencing have begun to reveal gaps in this paradigm. In particular these experiments have revealed that most genomes are pervasively transcribed, so that not only protein-coding genes but also many other regions of the genome are transcribed to produce so called non-coding RNA. As a result, DNA is often transcribed not only in the direction needed to make a protein (the sense direction) but also in the opposite (antisense) direction. The question then arises as to what all this extra transcription is doing: is it an accident, the inevitable by-product of the noisy cellular environment, or does it perform some regulatory function? Increasingly, the conclusion that it does have an important regulatory role is becoming accepted. However, the actual mechanistic role played by non-coding, often antisense, RNA is very unclear. The majority of previous studies have probed these questions at the level of a whole genome from which it is very difficult to draw conclusions about the regulation of specific genes. In this project, we propose to take a tightly focused look at antisense RNA regulation in the context of a plant flowering gene called FLC.FLC is a repressor of flowering, and is a gene whose quantitative level of transcription is vital in ensuring that the plant flowers at an optimal time for reproductive success. Under normal conditions the precise level of transcription is believed to be controlled by two antisense non-coding RNAs. Here, we are seeking to understand how the differential production of these two antisense RNAs is able to tune the expression of the FLC gene. One possibility that we will attempt to prove (or disprove) is that each individual FLC gene makes only one of the two antisense transcripts for extended periods of time and switches randomly back and forth between one state and the other over time. The two different states are believed to have very different effects on the expression state of the FLC gene. By controlling the length of time the system spends in one or other of the (bistable) states, the sense FLC expression level can then be precisely tuned. We will test this hypothesis (and others) to reveal in unprecedented detail how non-coding RNA works as a quantitative regulator of expression. If we can get to the heart of this mechanism we will have moved closer to answering our starting question: how is gene expression controlled?