Subproject C03: Molecular integration of light- and sugar-regulated alternative splicing

Principal investigator:

Dr. Andreas Wachter

Universität Tübingen

ZMBP, Allgemeine Genetik

Auf der Morgenstelle 28, 72076 Tübingen

Tel 07071 - 29 76149

Fax 07071 - 29 5042

andreas.wachterspam prevention@zmbp.uni-tuebingen.de

Summary:

Plants have evolved multiple and linked signalling pathways to sense ambient light conditions and their metabolic status, in order to adjust growth and other developmental processes to this critical information. We have demonstrated that photomorphogenesis, activated by exposing etiolated seedlings to light, is accompanied by widespread and specific changes in alternative precursor mRNA (pre-mRNA) splicing (AS) in Arabidopsis thaliana. Most AS events displayed a switch from a presumably unproductive variant in darkness to a protein-generating transcript in light, thereby activating the expression of selected genes upon illumination. For example, light-induced AS was shown to trigger expression of the potential splicing regulator RRC1 (REDUCED RED-LIGHT RESPONSES IN CRY1CRY2 BACKGROUND1), which regulates light-responsive hypocotyl growth. Studying the upstream regulatory mechanisms, we observed that light and sugar supply trigger similar AS changes in etiolated seedlings. Moreover, the major blue and red light photoreceptors were dispensable for the AS shifts upon white light, and played no or only a minor role in AS induced by illumination of etiolated seedlings with blue or red light. Based on the promptness of the AS shifts and the involvement of kinase signalling, we propose that light and sugar signals are sensed at the metabolic level to trigger phosphorylation-dependent changes in the activity of splicing regulators. In line with this hypothesis, we identified a group of splicing regulators that is subject to rapid and specific phosphorylation upon exogenous sugar supply. In the course of the project proposed here, we will test the specific role of candidate kinases, such as the central energy sensor SnRK1, in phosphorylating splicing regulators and regulating AS in response to altered metabolic signalling. Profiling phosphoproteomes in etiolated wild type and kinase mutant seedlings kept in dark or exposed to sugar/light will enable us pinpointing the splicing regulators that are targeted by kinase signalling under these conditions. We will test these splicing regulators’ contribution to sugar-/light-dependent AS and investigate the functional consequences of changes in their phosphorylation status, with a particular focus on their subcellular localisation patterns. Additional trans-acting factors and the cis-regulatory RNA motifs required to trigger specific AS changes in response to altered light and metabolic conditions will be identified and their interplay will be scrutinized. Our work will provide novel insight into the molecular mechanisms orchestrating specific AS programs linked to the metabolic status and light conditions, as part of gene regulatory responses that implement complex developmental transitions in plants.