Plant Alternative Splicing and Abiotic Stress

Alternative splicing is a genetic program that can either enlarge the potential of genes to encode for more than one protein or change the expression level of a gene. In this project we have characterized alternative splicing in plants. We show that it is much more prevalent than originally anticipated and that it greatly affects the expression program of genes important for plant development and for stress responses. In particular, we have defined an alternative splicing event where the sequences which are spliced out are in fact coding for protein parts and termed them exitrons (exonic introns). Our analysis exemplifies the importance of exitron splicing for plants as well as humans. Evolutionary analysis shows that exitron splicing evolved through a mechanism of intron loss followed by the evolution of splice sites preferentially in regions where there have been alternative splicing events occurring before. The overall aim of this project has been to address the essential dimension of alternative splicing in plants as a potential source of phenotypic variation by applying genomics technologies to provide new tools for discovery and quantitative analysis of alternative transcripts. With these tools we aimed to uncover the consequences and extent of alternative splicing. Together with our collaborators from Dundee, Posznan and Rehovot we have assessed high-throughput tools and techonolgies for the discovery and quantification of alternative splicing events in plants and have developed a sensitive RT-PCR platform and an RNAseq pipeline. Our project part decribes that in Arabidopsis more than 60 % of multi-exonic genes are alternatively spliced and that we have identified biological examples how this influences important regulatory genes. We also established rules for alternatively spliced transcript to be targeted by NMD (nonsense mediated decay), an important RNA degradation pathway. Interestingly, intron retention transcripts were not degraded by NMD although they had all the features for being a target for degradation. We show that these transcripts are localized to the nucleus and are therefore resistant to this RNA degradation pathway. In addition, by analyzing all intron retention events, we found a cohort of introns which surprisingly code for proteins and were therefore termed exitrons (exonic introns). In contrast to transcripts with classical intron retention events, the exitron containing transcripts have other characteristics and different fate and functions. Exitron splicing is regulated across tissues, in response to stress and in carcinogenesis. Altogether, our studies show that alternative splicing is as important in plants as has been shown for animals and that exitron splicing is a conserved strategy for increasing proteome plasticity in plants and animals.