In-depth functional characterization of chloroplast-localized mTERF proteins

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Organellar gene expression (OGE) is crucial for plant development, but the mechanisms that control it are still largely elusive. Thus, OGE requires various nucleus-encoded proteins that promote transcription, splicing, trimming and editing of organellar RNAs, and regulate their translation. Members of the so-called mitochondrial transcription termination factor (mTERF) family are found in metazoans and plants and regulate OGE at different levels. However, we are just beginning to understand the mechanistic details of their specific functions in these processes. In our preceding and preliminary work program we focused on the identification and selection for 10 mTERF proteins presumably functioning in the chloroplast. One additional mTERF, mTERF6, was characterized in detail. The mTERF6 protein is localized in chloroplasts and mitochondria and its knockout results in seedling lethality. We revealed a specific interaction between mTERF6 and a RNA sequence in the chloroplast isoleucine tRNA gene (trnI.2). However, mTERF6 can also bind to DNA of the same sequence. In vitro, recombinant mTERF6 bound to its plastid DNA target site can terminate transcription. In vivo, mTERF6 acts as a factor promoting chloroplast tRNAIle(GAU) (trnI.2) maturation. The major goals of our project are (i) to understand the molecular function of mTERF6 in detail, and (ii) to reveal the molecular functions of the remaining not yet investigated putative chloroplast-localized mTERF proteins. More specifically, we want to investigate the aminoacylation status of further organelle tRNAs in the leaky mterf6-1 mutant, and we want to determine the post-transcriptional modifications of trnI.2 with RNA mass spectrometry. In a second step, we will identify mTERF6 interaction partners by a combination of pull-down and guilt-by-association approaches. To reveal a possible involvement of mTERF6 in other transcriptional processes, RNA-Seq will be applied. Furthermore, the 10 selected mTERF proteins will be analyzed in more detail; in part by recruiting the methods successfully established to characterize the mTERF6 protein. To this end, we will apply a wide range of techniques that include biochemistry, mutant analysis, RNAi and protein over-expression, GFP localization studies, and stress and acclimation treatments. For the most promising mTERFs, RNA mass spectrometry, and RIP- and DIP-Seq analyses will lead to an in-depth characterization of their molecular functions.