Identification and characterization of RNA cis-elements and nucleases required for 5' processing of mitochondrial RNAs in Arabidopsis thaliana

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In Arabidopsis thaliana, the 5' ends of most mitochondrial RNAs are generated by post-transcriptional processing. We have identified a number of RNA PROCESSING FACTORS (RPF) required for the efficient formation of the 5' termini of several RNAs. These factors are pentatricopeptide repeat (PPR) proteins, which consist of canonical P motifs and lack enzymatic activity. Many of these factors are RESTORER of FERTILITY-LIKE proteins indicating the close mechanistic relationship between 5' processing and fertility restoration during breeding with cytoplasmic male sterile lines. RNA processing factors specifically interact with their target RNAs most likely recruiting a ribonuclease to the cleavage sites. The RNA binding sites can be predicted on the target RNA, but no experimental data have been obtained on the exact location and the structure of these cis-elements. In addition, virtually nothing is known about the postulated ribonuclease participating in 5' processing. In the proposed project, we will focus on these unsolved issues of post-transcriptional 5' processing in plant mitochondria.In the proposed project we will characterize the cis-elements for RPF3 and RPF6. Both factors are required for processing of ccmC transcripts derived from two different mitochondrial ccmC genotypes. These genotypes are differentiated by a 66 bp DNA segment, which contains mitochondrial sequences essential for processing. The 66 bp DNA segment is located upstream of the 5' termini that differ between ccmC transcripts derived from the two mitochondrial genotypes. Photoactivatable-ribonucleoside-enhanced crosslink-immunoprecipitation (PAR-CLIP) experiments and electrophoretic mobility shift assays with affinity purified recombinant RPF3 and RPF6 will exactly map the cis-elements and identify nucleotides involved in binding of these factors. The second topic will focus on two potential mitochondrial nucleases (MNU1 and MNU2) involved in 5' processing of at least some of the mitochondrial transcripts. These studies include RNase activity tests with recombinant proteins. In vivo investigations with epitope-tagged variants of MNU1 and MNU2 will give further information about the function of these proteins and individual domains thereof. Furthermore, the interaction of both proteins with each other and the interaction of MNUs with other proteins will be analyzed. Finally PAR-CLIP experiments will identify the RNA binding sites for MNU1, MNU2 and RPF2. RPF2 cooperates with MNU1 and MNU2 in the 5' processing of nad9 mRNAs.The proposed project will substantially contribute to our understanding of 5' processing of plant mitochondrial transcripts and will also shed light on the related processes involved in fertility restoration of cytoplasmic male sterility, an economically important mitochondrial trait used for the production of F1 hybrid seeds.