During the project we have pioneered state-of-the-art RNA sequencing (RNA-Seq) on nanopores to study complexity of pre-mRNA processing in Arabidopsis (Parker&Knop et al., 2020, eLife; Parker et al., 2020, biorXiv). This approach allowed us to resolve the complexity of mRNAs produced in the cell, including rare RNA isoforms of previously understudied immunity genes. Based on the nanopore RNA-Seq data we also generated the first global map of m6A positions. We validated this approach by sequencing all m6A-modified mRNAs fished out from the cell using an antibody recognising m6A (miCLIP) (Parker&Knop et al., 2020, eLife). Parallel analysis of control plants and mutant plants with decreased level of m6A, helped us to recognise an important role of m6A in transcription termination and mRNA stability. We have found that lack of m6A in pre-mRNAs results in the generation of shorter RNA isoforms. Some of these shorter RNAs can be rapidly degraded before being translated into proteins. Among the genes with expression levels affected by m6A we have found e.g circadian clock and immune-response genes, indicating the importance of m6A for plant development and stress response.
We next characterised the role of the RNA-binding protein FPA in regulation of expression of plant immune genes (Parker&Knop et al., 2020, bioRxiv). We have found that FPA is present in Pol II complexes during transcription termination and predominantly causes generation of shorter RNAs. The activity of FPA at many immune response genes leads to formation of prematurely terminated RNAs, that in turn results in production of non-functional proteins and increased susceptibility to pathogen attack. Even though FPA does not change global m6A levels in plants, it affects transcription termination at hundreds of genes, that, in turn, can affect m6A localisation, and thus function, in the cell.
We have also identified a novel factor involved in m6A regulation in Arabidopsis (Parker et al., 2021, manuscript in preparation). We have characterised the mutant plant that disrupt the function of this factor and found more evidence for m6A-dependent transcription termination. Further experiments are planned in order to understand the major role of this factor in gene expression regulation in plants.
Results generated in the course of the project have been presented to the broader scientific audience during national and international conferences (e.g. RNA UK 2021, RNA Meeting 2021) and have been discussed during local seminars and scientific meetings (e.g. group, institute and division seminars). We have already published one scientific paper containing data obtained within the project in an open access journal eLife (Parker&Knop, 2020, eLife). Another two papers have been recently published in bioRxiv (Parker et al., 2020, bioRxiv; Parker&Knop et al., 2020, bioRxiv), a free online archive and distribution service for unpublished preprints in the life sciences field. The corresponding manuscripts are in review in Genome Biology and eLife, respectively. Further scientific papers are in preparation. All data generated within the project are deposited in the publicly available repositories.
