Tuning gene expression through antisense transcript dynamics

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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?