Structure-based functional analysis of the Arabidopsis thaliana RNA editing factor MORF1
FacultiesFakultät für Naturwissenschaften
InstitutionsInstitut für Molekulare Botanik
RNA editing is one of the co- and posttranscriptional modifcations necessary for RNA maturation. Alongside polyadenylation, five-prime capping and splicing, organelles of embryophytes alter cytidines to uridines via deamination. This deamination reaction is mediated by a flexible protein complex called editosome. Editosomes in Arabidopsis thaliana contain Pentatricopeptiderepeat (PPR) proteins specifically recognizing cis-elements upstream of the respective editing site and potentially recruiting additional trans-factors. These trans-factors include the multiple organellar RNA editing factor (MORF) protein family, which is required for RNA editing in mitochondria and chloroplasts. Previously it has been shown that MORFs are able to both dimerize and to interact with PPR proteins. However, how these interactions occur had been unclear due to the lack of structural information about MORF proteins. Recently identified crystal structures of MORF proteins paved the way for more detailed interaction analysis and their functional relevance in vivo. In this thesis MORF1 structure based mutants were analyzed in yeast two-hybrid and pulldown studies as well as in in planta complementation assays in the EMS mutant line morf1-1 and the T-DNA insertion line morf1-2. The interaction studies indicated the necessity of a hydrophobic region mainly consisting of residues located in β-sheet 5 for MORF dimerization. On the other hand, PPR-MORF interactions were impaired not only by mutations within the hydrophobic region but also by mutations located at the N-terminus. Furthermore, constructs harboring mutations of L164, located in the hydrophobic region, were not able to complement the growth phenotype of morf1-1 plants as well as their RNA editing phenotype at several probed editing sites. In contrast other constructs with mutations located at the N-terminus and the hydrophobic region were able to rescue both the macro and RNA editing phenotype. Constructs with a mutation from L164 to glutamate also did not complement the morf1-2 embryolethal phenotype while mutation to alanine and constructs mutated at C85 did. Hence L164, necessary for MORF dimerization, is likely to be crucial for the function of MORF1. Moreover, this data suggests that specific MORF dimerization is crucial for the RNA editing machinery in plant organelles.
Subject HeadingsSchmalwand <Arabidopsis> [GND]
RNA editing [LCSH]