RNA-Metabolism in Mitochondria of Higher Plants
Natural genetic variation affecting mitochondrial transcripts
Natural genetic variation is observed between subspecies, varieties, lines and individuals of a given species. In Arabidopsis thaliana, substantial natural genetic variation exists between different accessions (ecotypes), predominantly geographically isolated inbred populations. Natural genetic variation can affect all kinds of traits including the structure or abundance of RNA. In comparative studies of 5' and 3' extremities of major mitochondrial RNAs a numbers of 5' end polymorphisms are observed among several accessions. Their appearance in F1 reciprocals revealed that theses polymorphisms can be attributed to differences in the mitochondrial DNA or in the nuclear genome (further details see Forner et al., 2008, Stoll et al., 2013). These polymorphisms are used to identify nuclear genes required for 5' processing of mitochondrial transcripts.
Identification and characterization of proteins required for posttranscriptional maturation of mitochondrial-encoded RNAs
Based on natural genetic variation affecting 5' ends of mitochondrial RNAs we used a linkage analysis to identify the nuclear genes required for 5' processing of various mitochondrial transcripts in Arabidopsis thaliana (Hölzle et al., 2011; Jonietz et al., 2010; Jonietz et al., 2011; Hauler et al., 2013; Stoll et al., 2014; Stoll et al., 2015). These factors, called RNA PROCESSING FACTORs (RPF), belong to the large family of pentatricopeptide repeat proteins (PPRP) with functions in the RNA metabolism in mitochondria or (/and) chloroplasts. Some of the RPFs show high similarity to a subgroup of PPR proteins that can restore male fertility in cytoplasmic male sterile lines in other plant species. In Arabidopsis thaliana, where cytoplasmic male sterility has not been described, this class of about 27 proteins acts as specificity factors enhancing endonucleolytic generation of mature 5' ends. RPFs most likely bind specifically to the one or several target RNA directing an endonuclease to the cleavage site. Whether this requires a direct interaction between the different proteins is unclear.
Branched-Chain Amino Acid Metabolism
The focus of our studies of branched-chain amino acid metabolism is dedicated to biosynthesis and degradation of these compounds in Arabidopsis thaliana. In this context we investigated BRANCHED-CHAIN AMINOTRANSFERASE 4 (BCAT4) a promiscuous enzyme with a major function in the methionine elongation pathway the first part of the biosynthesis of aliphatic glucosinolates. Methionine elongation and Leu biosynthetic pathways share identical reaction types suggesting a close evolutionary relationship between these reaction cascades. Apart from BCAT4, which catalyzes the committed step in the biosynthesis of aliphatic glucosinolates, BCAT3 and large (IPMI LSU1) and small (IPMI SSU2 and 3) subunits of isopropylmalate isomerase (IPMI) are involved in biosynthesis of branched-chain amino acids and of aliphatic glucosinolates or exclusively in the biosynthesis of these secondary metabolites (further reading: Schuster et al., 2006 and Binder, S. 2010).
Presently several members of the branched-chain aminotransferase gene family and different subunits of isopropylmalate isomerase are under investigation.
CRISPR-Interference (CRISPRi) and CRISPR-Activation (CRISPRa)
CRISPR–Cas (Clustered regularly interspaced short palindromic repeats- CRISPR associated) is an RNA-mediated adaptive immune system found in bacteria and archaea. In these prokaryotic organisms, CRISPR-Cas systems protect the cells from invasion by foreign DNA elements like phages or plasmids. The simplest version among many others is the type II CRISPR-Cas9 system, which is commonly used in genome editing in many different organisms. Essentially it needs only two components: the Cas9 endonuclease and the single guide RNA (sgRNA), which is engineered by the fusion of the CRISPR-RNA (crRNA) and the trans-activating CRISPR-RNA. But the CRISPR-Cas9 system can be also used to manipulate transcription. In this case a deactivated Cas9 (dCas9) protein is used that functions as a sequence-specific DNA binding protein. The dCas9 is fused to activator or repressor domains, to activate (CRISPR-Activation (CRISPRa)) or down regulate transcription (CRISPR-Interference (CRISPRi)). These dCas9 fusion proteins are directed to the promoter sequences of the target gene by multiple sgRNAs.
In a project funded by the Baden Württemberg-Stiftung, we try to establish orthogonal CRISPR-Activation (CRISPRa) and CRISPR-Interference (CRISPRi) to manipulate the metabolism in plants.