Several proteins form variable RNA editing complexes in plant organelles

Abstract

In RNA editing in plant organelles, PPR (Pentatricopeptide Repeat) proteins with an E or E and DYW domains at the C-terminus are important participants for recognizing the specific cis-elements. These proteins are able to connect to their target RNA sequence via the PPR domains. Recognition of the specific target RNA sequence by PPR proteins is based on the specific binding of one PPR motif to one nucleotide. There are several possibilities for the function of the E-domains in the PPR RNA editing factors: they may bind to the RNA as well as the PPR motifs, or provide the interacting surface for other proteins involved also in the RNA editing process. It is also not excluded that E domains perform these two possible tasks simultaneously. The DYW domain contains a conserved motif (HxExnCxxC) of cytidine deaminases and shows zinc-binding capability, suggesting the domain works as the so far missing deaminase enzyme. But its cytidine deaminase activity has not yet been proofed. The MORFs (Multiple Organellar RNA Editing Factors) also termed as RIPs (RNA editing factor Interacting Proteins) are also involved in several C-to-U substitution events in flowering plants mitochondria and/or in chloroplasts. Nine members of this family contain the conserved 100 amino acids long MORF box and one protein contains only the C-terminally half of that. The exact function of these proteins is unknown yet, but it is suggested that they work together with PPR proteins to perform certain RNA editing events maybe through their direct connections to PPR proteins at each MORF box. To understand further detailed functions of the three domains PPR, E and DYW in RNA editing factors, I employed three different approaches. The first one is functional complementation of the mef28-1 RNA editing mutants with chimeric PPR type RNA editing factors. The MEF28 is involved in the editing of two neighboring cytidines at the nad2-89 and 90 sites. We are interested in how this rarely event is performed by a single PPR protein. The DYW domain of MEF28 is not able to substitute to other DYW domains to edit the downstream cytidine in the two nad2 sites, suggesting its requirement for the flexible targeting function. In the second approach I used Y2H analyses for mapping the binding sites between MEFs (Mitochondrial RNA Editing Factors) and MORFs. In the most cases the bait wild type and partial MEF proteins could connect to the MORF1, MORF2, MORF8 and MORF9, rarely with MORF3 and very rarely with MORF4, MORF5, MORF6 and MORF7. In contrast, the wild type and the partial MEF8 bait constructs (except the MEF8_ECE+) could bind to almost all MORF preys, indicates that MEF8 is a very unique PPR protein. After Y2H analyses of several different partial E domain constructs, I found that the MEF21 is able to bind with the MORF1 through the N-terminal part of the E domain. Finally, I established the Pichia pastoris expression system to express recombinant PPR type RNA editing proteins. The system is now available for further molecular functional analysis of PPR proteins that have been difficult to be expressed in E.coli system.