Role of the RNA silencing machinery in the regulation of FLC.

The Dean laboratory is interested in understanding what controls the timing of flowering in a wide range of plants. We are using a model plant Arabidopsis thaliana to identify all the genes involved in controlling flowering as in this plant it is easy to clone the gene based on just having a mutation (a lesion) in that gene and no other information. Over the years we have shown that a key player in the regulation of flowering is a protein called FLC. FLC prevents flowering by stopping the activation of a set of genes needed to make flowers. FLC levels are reduced by a winter period which is why many plants flower in the spring. It is also reduced by a set of genes grouped together in the so-called autonomous promotion pathway. We have recently also found FLC levels are regulated by a new gene regulatory pathway involving very small RNA molecules. Studies on virus resistance in plants and development in worms has revealed that RNA molecules between 21 and 24 nucleotides long act as messengers to zap longer RNA molecules containing the same sequence. They also get embedded in large protein complexes and guide them to the genetic material in the cell (chromatin = DNA looped around proteins called histones) finding DNA with a matching sequence. The processes involving small RNAs have been called RNA silencing pathways and are used in human therapeutics to switch off genes under the name RNAi or RNA interference. How RNA silencing is activated and how it leads to regulation of the chromatin structure of regular genes is not known. We want to dissect these processes by analysing FLC regulation. This system is likely to produce concepts relevant to gene regulation throughout biology.

The role of the RNA silencing machinery in RNA-mediated transcriptional repression of repeated sequence DNA and transposons is beginning to be understood but its importance for regulation of single copy endogenous genes has yet to be explored. For a number of years, we have been analysing the different pathways that control expression of FLC, a floral repressor that regulates the key developmental switch to flowering in Arabidopsis. We have recently found that FLC is mis-regulated in mutants defective in RNA silencing. We will determine which RNA silencing components regulate FLC in different tissues and at different stages of development and undertake a genetic analysis to investigate if the RNA silencing activities are integrated with other pathways regulating FLC, focusing on the autonomous promotion pathway. We have detected small RNAs homologous to the 5' and 3' regions of FLC and antisense transcripts differing in abundance in different tissues have been found in whole genome array analyses. We will characterize these small RNAs and antisense transcripts further and investigate their production in different RNA silencing and autonomous pathway mutants. We will also investigate whether the RNA silencing machinery functions downstream of two of the components of the autonomous promotion pathway FCA and FY, identify other components required for their repression of FLC and investigate the role of FCA and FY and the RNA silencing components in FLC chromatin regulation. This will test the model that FCA/FY through changes in the processing of FLC nascent transcript, initiate a series of events involving small RNA production, RNA-guided recruitment of chromatin complexes, modification of the histones at FLC and transcriptional repression.