// P06
Alternative mRNA isoforms generated in the nucleus as a source for organellar adaptation to stress
Project Leader// Prof. Dr. Sascha Laubinger
Martin-Luther-University Halle-Wittenberg
Institute of Biology
Department of General Genetics
Background
Plants possess a remarkable ability to adapt to environmental stresses, and one key mechanism driving this flexibility is the generation of alternative mRNA isoforms. By utilizing different transcription start sites (TSS), transcription termination sites (TTS), and alternative splicing events, plants can expand their proteome diversity, creating functionally distinct protein isoforms. Our lab focuses on how these alternative mRNA isoforms are generated and how they contribute to plant stress adaptation. Organelles play a central role in plant stress responses by coordinating metabolic and signaling pathways. Stress-induced alternative mRNA isoforms can influence organelle function by modulating the composition of organelle-targeted proteins, potentially affecting key adaptive processes
Our Project in RTG2498
In this project, we put particular emphasis on nuclear-encoded genes producing mRNA isoforms, the products of which can function in different plant organelles. We have used various RNA sequencing techniques to detect stress-induced mRNA isoforms in plants subjected to environmental challenges such as salt stress and drought stress. To deepen our understanding, we employ an integrative approach combining cell biology, biochemical methods, and genetic analysis:
- Identifying Alternative mRNA Isoforms Under Stress
We previously used CAGE-seq and direct RNA-seq data from Arabidopsis plants grown under stress and non-stress conditions to detect changes in TSS usage and alternative splicing patterns. We found hundreds of candidate genes that produce alternative isoforms upon stress and encode proteins targeted to different organelles. Targeted expression analysis and the dynamics of candidate genes will be further assessed. - Tracking Protein Localization
To determine whether alternative mRNA isoforms result in proteins targeted to distinct cellular compartments, we will conduct transient expression experiments in Nicotiana benthamiana. By fusing candidate mRNA isoforms with fluorescent protein markers, we visualize their subcellular localization and narrow down the most functionally relevant isoforms for further investigation. - Functional Characterization of Alternative Protein Isoforms
We will generate transgenic Arabidopsis lines overexpressing specific isoforms, complemented by knockout mutants created via CRISPR or T-DNA insertion lines. These plants will undergo phenotypic analysis under stress conditions to assess the impact of specific isoforms on plant resilience. Functional validation includes complementation assays, where knockout mutants are rescued with constructs expressing only one specific mRNA isoform, ensuring precise functional insights
Why It Matters
By uncovering how alternative mRNA isoforms fine-tune plant responses to stress, we aim to reveal novel regulatory mechanisms with broad implications for plant stress resilience. Understanding the functional consequences of mRNA isoform diversity will pave the way for innovative strategies to enhance plant stress tolerance in the face of global challenges.
