Periodic Reporting for period 1 - PLANT-RNA-MET (A newly discovered role for mRNA methylation in controlling plant gene expression)
Berichtszeitraum: 2018-03-01 bis 2020-02-29
The ability of plants to grow, develop and respond to the environment depends upon the ability to precisely co-ordinate gene expression. As development proceeds, or defences to pathogens are mounted, the expression of some genes needs to be silenced, while other must be activated or amplified. Therefore, explaining how gene expression is regulated is important to understand how plant development and immunity are controlled. In many aspects, the principles of gene expression regulation in plants resemble that of other complex organisms, like humans or animals.
Genes are segments of DNA, that during gene expression are transcribed by an enzyme called RNA polymerase II (Pol II) complexed with other components, into precursor messenger RNA (pre-mRNA). The efficiency of transcription depends on many factors, e.g DNA modifications that make DNA accessible or inaccessible to Pol II complexes. In the next step, pre-mRNA is processed in many ways into mature form - messenger mRNA (mRNA), that can affect mRNA fate in the cell. For example, the same pre-mRNA can be processed into mRNAs that differ in the position at which they end as a result of a process known as alternative polyadenylation. Since mRNA is next translated into protein, differences in pre-mRNA processing can determine what protein the gene will code for or what the lifetime of mRNA will be. Associated with pre-mRNA processing, is a newly recognised layer of gene regulation involving RNA modifications. For example, m6A RNA methylation is essential for regulation of mRNA fate, including mRNA degradation and translation efficiency. Our understanding of the diverse functional impacts of m6A in plants is still emerging.
In this proposal, we aimed to explain the function of m6A mRNA modification in plant gene expression, with the major focus on plant immune response genes. To do this, we designed our experimental plan to recognise how m6A affects transcription termination and alternative polyadenylation, and to identify important factors involved in this regulation. Arabidopsis thaliana was used in this study because it is a model experimental system. A better understanding of the role of m6A in tuning gene expression in Arabidopsis facilitates the new knowledge and understanding required for the development of crops with improved growth and immunity against pathogens.
Genes are segments of DNA, that during gene expression are transcribed by an enzyme called RNA polymerase II (Pol II) complexed with other components, into precursor messenger RNA (pre-mRNA). The efficiency of transcription depends on many factors, e.g DNA modifications that make DNA accessible or inaccessible to Pol II complexes. In the next step, pre-mRNA is processed in many ways into mature form - messenger mRNA (mRNA), that can affect mRNA fate in the cell. For example, the same pre-mRNA can be processed into mRNAs that differ in the position at which they end as a result of a process known as alternative polyadenylation. Since mRNA is next translated into protein, differences in pre-mRNA processing can determine what protein the gene will code for or what the lifetime of mRNA will be. Associated with pre-mRNA processing, is a newly recognised layer of gene regulation involving RNA modifications. For example, m6A RNA methylation is essential for regulation of mRNA fate, including mRNA degradation and translation efficiency. Our understanding of the diverse functional impacts of m6A in plants is still emerging.
In this proposal, we aimed to explain the function of m6A mRNA modification in plant gene expression, with the major focus on plant immune response genes. To do this, we designed our experimental plan to recognise how m6A affects transcription termination and alternative polyadenylation, and to identify important factors involved in this regulation. Arabidopsis thaliana was used in this study because it is a model experimental system. A better understanding of the role of m6A in tuning gene expression in Arabidopsis facilitates the new knowledge and understanding required for the development of crops with improved growth and immunity against pathogens.
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.
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.
We were the first to globally map m6A in any species using RNA-Seq on nanopores and pioneered the use of this sequencing technology to understand the complexity of pre-RNA processing. This is important for understanding gene expression, not only in plants, but more widely. Our work with plant immune-response genes reveals a previously unrecognised layer of regulation that has important implications for understanding how plants react to pathogens and how these responses evolve. Consequently, these basic findings inform rationale-based approaches to the future development of disease resistance crop plants.