DNA damage and alternative splicing in plants

There is an increasing evidence that post-transcriptional processes play an important role in gene expression regulation. Most messenger RNAs in higher eukaryotes are synthesized as precursors which contain intervening sequences, termed introns. Differential intron removal and splicing of the exons by alternative splicing (AS) may result in distinct protein isoforms or affect mRNA stability thus affecting transcriptome and proteome diversity in eukaryotic cells. Ser/Arg-rich (SR) proteins, a family of evolutionary highly conserved splicing factors, are one of the determinants of splicing patterns present in different cells and at specific developmental stages or conditions. Arabidopsis genome comprises 18 genes coding for SR proteins which is nearly double the number found in humans. Plant-specific SR proteins are particularly interesting as they might have evolved to serve plant oriented needs. Current estimates of the occurrence of alternative splicing entirely change the way we think about gene expression regulation in plants. Approximately 60% of intron-containing genes in Arabidopsis are alternatively spliced under normal growth conditions. This number is probably higher as it does not capture AS events specific for various environmental conditions that plants experience through their life cycle. Both environmental stresses and endogenous processes can induce DNA damage and impair genome stability. Cell response to DNA damage must be tightly regulated to ensure efficient repair on one side, and to prevent unnecessary and potentially harmful effects during normal cellular processes on the other side. Research of DNA damage response (DDR) both in plants and animals has focused on the transcriptional regulation by transcription factors, signaling pathways mediated by kinases and phosphatases, and lately on epigenetic regulation. Recent studies in animals have unexpectedly revealed that splicing factors and alternative splicing are important players in the DDR. Despite extensive studies of DDR and DNA repair pathways carried out in plants regulation at the level of alternative splicing has not been comprehensively addressed. In this project we will investigate the link between DNA damage, DNA repair and alternative splicing so far never explored in plants. Specifically, we will identify transcriptional and AS changes in response to DNA damage in Arabidopsis at the genome wide level. We will identify in vivo binding motifs and direct RNA targets of a plant- specific SR protein involved in AS regulation of DNA repair. We will perform small scale comparative studies in the moss Physcomitrella patens to gain evolutionary insights into the regulation of DNA damage response by SR proteins. Our project will contribute to understanding how alternative splicing can complement transcriptional/post- translational regulation of DNA repair and genotoxic stress response in plants.

Since the conquest of land, plants, as sessile organisms, have adapted to a multitude of varying environments. It is this sessile nature, which calls for well-orchestrated stress response and gene expression regulation, as both natural and agricultural environments hold various sources of biotic and abiotic stressors that may result in DNA damage. Besides exposure to environmental stress, several cellular processes yield a potential threat to DNA integrity. It is therefore essential to understand what strategies plants have developed for DNA damage detection and repair. Alternative splicing, a process that allows one gene to produce more than one protein variant and also influences protein abundance and availability, is an important player in gene expression regulation. Recent studies, including ours, demonstrated that the majority of plant genes are alternatively spliced, highlighting the importance of alternative splicing in plant development and stress response. To further advance our knowledge on alternative splicing in plants, we developed methods for its improved detection and quantification. We show that ~34,000 genes produce more than 82,000 RNA variants in the model plant Arabidopsis thaliana. Strikingly, we have discovered an overlooked alternative splicing event, which we named exitron splicing. Exitron splicing increases the number of protein variants produced by a single gene and influences protein function. At least 6.6% of Arabidopsis and 3.7% of human protein-coding genes contain exitrons. Exitron splicing occurs in many essential plant and human genes, including those involved in stress response, DNA damage repair, and cancer. We could identify multiple Arabidopsis genes, which change their alternative splicing in response to DNA damage. We have investigated the role of splicing regulators, which control this change. We were particularly interested in the function of plant-specific splicing regulators as they might have evolved to serve plant-specific needs. We could show that they themselves are regulated in response to stress and DNA damage, and this regulation depends on the light conditions. Remarkably, this regulation is conserved in Arabidopsis and the moss Physcomitrella patens, species that diverged more than 400 Mio years ago. Further, by analyzing the mutant and overexpression lines of these splicing regulators under various growth conditions, as well as splicing patterns of their target genes, we have identified responses to light/dark and DNA damage as highly conserved expression modules established before the divergence of flowering plants and mosses. These findings strengthen the importance of alternative splicing in gene expression regulation during plant growth and stress response.