Alternative splicing in plants: Regulation by SR proteins
(SFB projects 1710 /1711)

Splicing is a highly dynamic process as the spliceosome has to assemble anew on every intron. In addition, during splicing the spliceosome rearranges to accommodate for the two transesterification steps and this process has been shown to be accompanied by changes in protein composition. Usage of alternative splice sites has expended the repertoire of gene expression and has been exploited for various differentiation processes. In plants, the recognition of an intron varies somehow from that in animals and is therefore the focus of our investigations. To this end, we have isolated and partially characterized about nineteen Arabidopsis proteins mostly belonging to the family of Arg/Ser-rich splicing factors whose members are important for splice site selection, spliceosome assembly and various developmental processes. In addition, we have isolated and partially characterized several regulatory proteins which seem to be essential to drive the splicing process, like SRPK kinases, helicases and cyclophilins.
Determining interaction partners of plant SR proteins: In order to investigate the impact of SR protein on plant development and on the response to environmental cues, we are performing several approaches to find binding partners for plant SR proteins. Protein-protein interaction partners of selected SR proteins (RSZ33) are investigated by a yeast-two hybrid screen and by isolating TAP-tagged complexes. RNA targets of SR proteins are determined by CLIP (Cross-linking and immunoprecipitation ) and a genomic SELEX methods. The CLIP method is currently tested in the S. pombe system and will then be developed for Arabidopsis. In addition, expression analysis by microarray gene chips and Arabidopsis tiling arrays are being performed. In the framework of an European Network of Excellence on Alternative Splicing (EURASNET, www.eurasnet.info/) we have established together with two other plant groups (J. Brown, Dundee, A. Jarmolowski, Poznan) an medium RT-PCR splicing panel for more than 400 alternative splicing events. The analysis of our mutant and overexpressing plants will contribute in defining pathways were these SR proteins are involved.
Evolutionary conservation of alternative splicing events in plant specific SR proteins: Our recent findings of evolutionary conserved sequences of alternative splice sites in the genes of the plant specific proteins atRSp31 and atRSZ33 led us to the questions why these alternative splicing events are so highly conserved and how these events are regulated. We are investigating the location and the impact of the stable alternative transcript of atRSp31 on gene expression and plant development. As conservation on the genomic sequence level is indicative of an RNA related mechanism, we are investigating these alternative splicing events in mutants of other RNA metabolic pathways (like siRNA, microRNA, tasiRNA, nonsense mediated decay (NMD) etc.). The moss Pyscomitrella patens is an ancestral land plant and a good system for analyzing evolution and redundancy of plant genes as it easy to obtain mutants by homologous recombination. We therefore study atRSp31 and atRSZ33 orthologues in this system and analyze the impact of knock-out and overexpression mutants. This will allow us to assess the conserved alternative splicing event in moss.