RNA-protein-protein interactions of plant RNA editing factors
Final Report Abstract
We have made progress in the identification and characterization of protein-protein interactions between various PPR-type RNA editing factors and MORF proteins with the support of funding through the present DFG project. RNA editing in plant organelles alters specific Cytidines to Uridines in mRNA, tRNA and rRNA. Among several types of RNA editing factors, PLS-type PPR proteins and MORF proteins are the two major key players for RNA editing in organelles of flowering plants. PPR-type RNA editing factors consist of a N-terminal PPR domain, which interacts with RNA in a sequence specific manner and a C-terminal E domain or E-DYW domain. The C-terminal DYW domain most likely has the enzymatic activity for the C to U conversion. MORF proteins are essential for more than 50 editing sites, in contrast to PPR proteins, which are specifically necessary for one or few sites. To understand the relevance of these two types of factors, several approaches to investigate protein-protein interactions have been developed. Among the various tested methods, we employed two relatively robust systems for routine protein-protein interaction analyses, Y2H and in vitro CoIP with multiple tags. The two procedures could be applied to almost all of the tested PPR-type editing and MORF proteins. MORF proteins showed specific and strong homo- and heteromer interactions but did not directly interact with RNA. Furthermore, we demonstrated that MORF proteins associate with PPR elements as well as E domains in the PPR-type editing factors. Generally, the affinity of MORF proteins to E domains of PPR proteins was stronger than to PPR elements. This observation suggests that the E domain-MORF interactions, which have not been included in the previous co-crystallization analysis, is particularly important for the RNA editing function. Since expression of full-length PPR or MORF proteins has been very difficult, we expressed fragments of MORF and PPR proteins for the functional investigation of each domain. MORF fragments containing conserved MORF domains were successfully expressed and their 3D structures were determined. This result also supported the strong and specific dimerization of MORF proteins as observed in Y2H and CoIP results and presented a strong hint that dimerization of MORF is a requirement for their function in plant organellar RNA editing.
Publications
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(2015) Selective homo- and heteromer interactions between the multiple organellar RNA editing factor (MORF) proteins in Arabidopsis thaliana. J. Biol. Chem., 290, 6445–56
Zehrmann, A., Härtel, B., Glass, F., Bayer-Császár, E., Obata, T., Meyer, E., Brennicke, A. and Takenaka, M.
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(2017) Crystal structures of the Arabidopsis organellar RNA editing factors MORF1 and MORF9. Nucleic Acids Res. 45, 4915–4928
Haag, S., Schindler, M., Berndt, L., Brennicke, A., Takenaka, M., Weber, G.
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(2017) The conserved domain in MORF proteins has distinct affinities to the PPR and E elements in PPR RNA editing factors, BBA Gene Regul. Mech. 1860:813-828
Bayer-Császár E., Haag, S., Jörg A., Glass F., Härtel, B., Obata, T., Meyer, E. H., Brennicke, A., Takenaka, M.
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(2018) The pentatricopeptide repeat protein MEF31 is required for editing at Site 581 of the mitochondrial tatC transcript and indirectly influences editing at Site 586 of the same transcript. Plant Cell Physiol. 59(2):355-365
Arenas-M A, González-Durán E, Gómez I, Burger M, Brennicke A, Takenaka M, Jordana X
