Regulation and functions of alternative splicing in plants
2 PhD projects offered in the IPP summer call 2023
Alternative splicing (AS) massively increases transcriptome complexity in higher eukaryotes, with critical implications in development and stress responses. Studying AS events in animals and plants revealed the existence of diverse regulatory principles, however, the mechanisms by which rapid and coordinated changes in the AS output can be achieved are still poorly understood. The Wachter group and others have previously shown that seedling photomorphogenesis is accompanied and supported by AS changes. Furthermore, splicing regulatory proteins involved in light-dependent AS and seedling development have been identified. In our ongoing research, we examine the molecular mechanisms and signaling pathways underlying rapid re-programming of the transcriptome via AS to steer seedling development in a light-dependent manner.
PhD Project 1: Regulation of alternative splicing by R-loop formation in Arabidopsis
R-loops refer to RNA-DNA hybrid structures that have been linked to genotoxic stress. More recently, R-loops have also been shown to function in the regulation of gene expression. A previous study generating a genome-wide R-loop atlas for the model plant Arabidopsis thaliana using ssDRIP-seq (single-strand DNA ligation-based library preparation from DNA:RNA hybrid immunoprecipitation followed by sequencing) identified major changes in R-loop formation in the comparison of dark- and light-grown seedlings (Xu et al., 2020). Furthermore, the Wachter group has demonstrated rapid re-programming of alternative splicing (AS) upon exposing etiolated seedlings to light (Hartmann et al., 2016 & 2018; Saile et al., in revision). Based on first reports connecting R-loop formation and AS (e.g., Conn et al., 2017), we would like to examine whether these two processes are more widely coupled. In the context of this PhD project and as part of the 4R RTG “R-loop Regulation in Robustness and Resilience”, we will address the following major questions: 1) Are R-loops overrepresented in regions displaying light-dependent AS? 2) Does light exposure of dark-grown seedlings alter R-loop formation as quickly as changes occur on the level of AS? Here, it is planned to test the effects of photosynthetically active white light and UV-B light, which can also cause genotoxicity? 3) Do mutants with altered expression of the nuclear RNH1A show alterations in their light responses, considering R-loop formation, AS, and development? Resolving these questions will also provide a basis to further study the mechanistic link between R-loops and AS at a later stage of the project.
This project will be part of the RTG on R-loop Regulation in Robustness and Resilience (4R), and is pending of this programme being supported by third party funding.
If you are interested in this project, please select Wachter (Rloop) as your group preference in the IPP application platform.
PhD Project 2: Condensation of RS splicing regulators in light-dependent plant development
We have previously demonstrated the occurrence and functions of light-triggered AS in etiolated seedlings (Hartmann et al., 2016 and 2018). Moreover, treatment with external sucrose triggered similar AS changes as illumination and a role of the central energy sensor kinases SnRK1 and TOR in this process was revealed (Saile et al., in revision). The rapid phosphorylation and condensate formation of RS proteins upon exposing etiolated seedlings to light or sucrose as well as altered photomorphogenesis of rs mutants suggest a key role of RS proteins in light-dependent development. In this project, we will use a combination of in vitro and in vivo experiments to study the molecular determinants and biological functions of RS condensates. The RS proteins consist of two RNA recognition motifs (RRMs) and an arginine/serine-rich domain predicted to be intrinsically disordered and responsible for condensate formation. Based on our preliminary work for recombinant production of RS proteins, we will generate domain mutants and study their condensation using turbidity and sedimentation assays. GFP-containing fusion proteins will be investigated with respect to droplet formation and dynamics. The most interesting RS protein versions will then be used to complement Arabidopsis thaliana mutants or generate overexpression lines to study RS functions and domain contributions in light-dependent speckle formation, AS, and seedling development. Assuming that phase separation of RS41 is linked to altered molecular interactions, we will identify proteins and RNAs that are in contact or in complexes with RS41, by comparing etiolated seedlings before and after light/sucrose exposure. These experiments will involve in vivo labelling techniques established in the Wachter group such as TRIBE for RNAs (targets of RNA-binding proteins identified by editing; Loeser et al., in revision) and proximity labelling for proteins. Candidate factors will be independently validated, e.g., by using iCLIP for RNA targets of the RS proteins, and will provide the basis for functional studies in follow-up projects. This project is part of the CRC 1551 (https://crc1551.com/) on “Polymer concepts in cellular function” and will be conducted in collaboration with the group of Prof. Friederike Schmid from the Physics department, who will be providing theoretical and computer modelling approaches.
If you are interested in this project, please select Wachter (RS) as your group preference in the IPP application platform.
Publications relevant to this project
Conn VM, Hugouvieux V, Nayak A, Conos SA, Capovilla G, Cildir G, Jourdain A, Tergaonkar V, Schmid M, Zubieta C and Conn SJ (2017) A circRNA from SEPALLATA3 regulates splicing of its cognate mRNA through R-loop formation. Nat Plants, 3:17053 Link
Hartmann L, Wießner T, Wachter A (2018) Subcellular Compartmentation of Alternatively Spliced Transcripts Defines SERINE/ARGININE-RICH PROTEIN30 Expression. Plant Physiology, 176:2886-2903 Link
Hartmann L, Drewe-Boß P, Wießner T, Wagner G, Geue S, Lee HC, Obermüller DM, Kahles A, Behr J, Sinz FH, Rätsch G, Wachter A (2016) Alternative Splicing Substantially Diversifies the Transcriptome during Early Photomorphogenesis and Correlates with the Energy Availability in Arabidopsis. Plant Cell, 28:2715-2734 Link
Saile J, Wießner-Kroh T, Erbstein K, Obermüller D, Pfeiffer A, Janocha D, Lohmann J, Wachter A.SnRK1 and TOR control light-responsive splicing events and development in etiolated Arabidopsis seedlings. in revision
Xu W, Li K, Li S, Hou Q, Zhang Y, Liu K and Sun Q (2020) The R-loop atlas of Arabidopsis development and responses to environmental stimuli. Plant Cell, 32:888–903 Link