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Understanding the Role of Antisense lncRNA in Vernalization, Memory and Life History

Periodic Reporting for period 1 - UNRAVEL (Understanding the Role of Antisense lncRNA in Vernalization, Memory and Life History)

Reporting period: 2016-04-01 to 2018-03-31

Optimal timing of flowering is of central importance to plant fitness, in both ecological and agricultural contexts. Plants integrate a variety of cues to determine the right time to flower; for many species, this includes an obligate requirement for the long period of cold experienced over winter (vernalization). In the face of an increasingly warm and variable climate, clarifying the mechanism of cold signal integration in determining flowering is of high importance.

In the model plant Arabidopsis thaliana, the integration of cold as a flowering signal is controlled by the flowering repressor gene FLOWERING LOCUS C (FLC). During winter, prolonged cold quantitatively down-regulates FLC expression across the whole plant, acting via an “ON/OFF” epigenetic switch at the level of the single cell. A long non-coding RNA, COOLAIR, is transcribed antisense to FLC and plays a key role in this switching mechanism. Plants in which COOLAIR transcription has been blocked show slower silencing of FLC in the cold and altered epigenetic switching dynamics. Like many non-coding RNAs in other systems, COOLAIR RNA can form folded secondary structures in in vitro systems that seem to be conserved across related species. This folding may be functionally important to the silencing of FLC in winter, perhaps by recruiting proteins known to be involved in the epigenetic switch.

Here, plants with slightly altered COOLAIR sequences were produced with the intention of disrupting their folded structures. Some of the alterations generated plants with late flowering and atypical patterns of FLC silencing in cold, suggesting a functional role for the folded structure. In parallel, ongoing work in the Dean lab and collaborators, methods were developed for determining the folded structures of COOLAIR in planta, and for identifying proteins that associate with the RNA during vernalization.
In collaboration with the Ding group at the John Innes Centre, methods were assessed for the probing and sequencing of COOLAIR under normal and vernalizing conditions to determine in vitro and in vivo RNA secondary structures. An approach to overcome low transcript numbers and isoform complexity, which complicated the subsequent sequencing of COOLAIR, has been determined and is in progress. COOLAIR RNA structure mutant plants and corresponding controls have been established and phenotyped, revealing late flowering, inhibited epigenetic modification changes and variable vernalization responsiveness; these lines are valuable resources for ongoing analysis of the functional consequences of RNA structure mutations. Surprisingly, no evidence was¬¬ found linking COOLAIR and the uncharacterised enzyme WLC in vernalization, despite apparent similarities in their epigenetic silencing functionality. ChIRP-MS has been developed within the Dean lab for the identification of COOLAIR:protein interactions, and preliminary results suggest candidate binding partners for follow-up work. While technical challenges have delayed completion, the research and collaborations established in the course of the Fellowship are ongoing. This research is enhancing our knowledge of the mechanism underlying ¬cold-mediated silencing in FLC specifically, and of the integration of long-term environmental signals in plants in general. More broadly, it contributes to our emerging understanding of the role of lncRNAs in an epigenetic silencing mechanism that is broadly conserved across the higher eukaryotes. Preliminary results have been shared in presentations both via group and institute presentations and internationally, and publication of results from this Fellowship is foreseen in late 2018/2019.
Through the course of this project, two main approaches represent progress beyond the state of the art: the ongoing development of methods to detect specific, extremely low copy RNA transcripts for structure probing in planta; and identification of RNA:protein interactions via mass spectrometry-based methods, which has not to date been published in plants. These methods, alongside the establishment of suitable COOLAIR structure mutant plants in which to test hypotheses, bring us closer to understanding of the role of RNA transcription and structure in epigenetic switching. In the longer term, clarifying the role of COOLAIR in vernalization in Arabidopsis will allow translation of this knowledge into crops, in particular economically important and closely related Brassica species. This ultimately facilitates targeted mining of germplasm for breeding more climate-resilient crops.

Alongside scientific outcomes, this project has also achieved Horizon 2020 Work Programme goals: developing creative potential and diversifying competences through international mobility and advanced training in technical and transferable skills; enhancing contact networks for both the Fellow and host organization through academic, commercial and public engagement; and catalysing significant career development. As a result, the Fellow has secured a permanent position in research in New Zealand. The research themes and collaborations established in the UK during the course of the Fellowship will be further developed in this new research role.