Composition and function of ribonucleoprotein complexes involved in chloroplast intron splicing

• Applicants: Dr. Jessica Jacobs; Professor Dr. Ulrich Kück
• Subject Area: Plant Genetics and Genomics
• Term: from 2012 to 2017
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 226524973

In the model alga Chlamydomonas reinhardtii, the mRNA of the chloroplast P700 chlorophyll a/b binding protein (psaA) is generated by trans-splicing of two group II introns. In this research project, we will investigate the composition and function of ribonucleoprotein complexes involved in chloroplast trans-splicing. In particular, this project is aimed to answer the following questions: 1. Can chloroplast spliceosomes be considered to be the precursors of nuclear spliceosomes?2. Is the composition of the organelle spliceosome dependent on environmental changes?The characteristics of group II introns suggest that they are ancestors of nuclear introns. They show similar boundary sequences and structural similarities and have identical splicing pathways. In vivo, the splicing of group II introns is dependent on several co-factors. We will investigate the contribution of co-factors on splicing of the tripartite psaA group II intron from the chloroplast psaA gene. Mainly genetic analysis has shown that 14 nuclear encoded proteins are essential for this trans-splicing process. These nuclear encoded components are involved in the splicing of the first or second psaA intron, or alternatively have a function in both splicing reactions. Some of these components are part of a high-molecular soluble or membrane-associated ribonucleoprotein complex. Previous experiments have identified two novel genes which encode proteins involved in trans-splicing. These proteins will be used as bait to identify protein-protein or protein-RNA interactions. With these experiments, we will define the composition of a chloroplast spliceosome. So far, no comparable complex has been identified from any eukaryotic organelle. We further will focus on the composition of the spliceosome which might be dependent on different environmental conditions. Our preliminary data indicate that under anaerobic growth conditions chloroplast spliceosomes in C. reinhardtii differ significantly in their composition. Finally, we will investigate the function of the identified trans-splicing factors. We propose that these components support the folding of the active intron secondary structure. Therefore, in vitro folding studies with different fragments will be conducted. Our studies will contribute to our understanding of the function of trans-splicing factors on intron folding.

• DFG Programme: Research Grants
• Participating Person: Professor Dr. Michael Schroda