Identification of nuclear encoded genes involved in 5' end processing of mitochondrial mRNA in Arabidopsis thaliana
Final Report Abstract
In Arabidopsis mitochondria, the post-transcriptional generation of 5' transcript ends is a frequent process. In the completed funding period, we identified four new, nucleus-encoded RNA PROCESSING FACTORs (RPF4 to RPF7) involved in this process. RPF4 and RPF6 as well as the previously described RPF1 to RPF3 belong to the group of RESTORER OF FERTILITY-LIKE (RFL) pentatricopeptide repeat (PPR) proteins. RFL genes exhibit an extraordinary high variation within a given species. Since genetic variation is a crucial prerequisite for our forward genetic approaches there is a bias towards the identification of RPFs within the group of RFL proteins explaining the overrepresentation of RFLs among the so far identified RPFs. RPF4 is necessary for the generation of ecotype-specific ccmB transcripts in the ecotype Ler. RPF4 in Ler and RFL9 in Col, alleles of the same gene, exhibit distinct ecotype-specific functions, which is consistent with the extreme variability of these genes and proteins. RPF3 and the now described RPF6 are required for processing ccmC mRNAs with two different 5' ends. These distinct ccmC transcripts are generated from RNA precursors transcribed from two different mtDNA configurations present in different ecotypes. There is a strict correlation of the two ccmC genotypes with the distinct transcript lengths and the specific functions of RPF3 and RPF6 in the generation of the different 5' termini. Thus two co-adapted cyto-nuclear systems correlate with the generation of two distinct ccmC transcripts. In contrast, RPF5 and RPF7 represent classical P-class PPR proteins but do not belong to the RFL group. RPF5 is crucial for 5' processing of nad6 mRNAs and of 26S rRNA. It exhibits also a minor influence on the 5' maturation of atp9 mRNAs. 5' un-processing 26S rRNA species exhibit a strongly reduced stability indicated by decreased accumulation to about 50% of the level of the mature 26S rRNA in wild-type plants. RPF7, required for efficient 5' processing of nad2 mRNAs, is a comparatively small protein comprising only eight PPR motifs. The small size of this protein suggests that another PPR protein might be involved in nad2 mRNA processing. The binding sites of various RPFs were predicted between about 20 and 130 nucleotides upstream of the potential cleavage sites. For RPF3 and RPF6, these potential recognition sites were confirmed with electrophoretic mobility shift assays. However, further detailed experimental studies are necessary to obtain a comprehensive view on the specific interaction between RPFs and their recognition sites in the RNA targets. Since RPFs do not have enzymatic activity, additional proteins with ribonucleolytic activity have to be involved in the cleavage 5' reaction and it will be highly interesting to unravel the mechanism of RPF-directed site-specific 5' processing of transcripts in plant mitochondria.
Publications
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(2013) Mitochondrial transcript length polymorphisms are a widespread phenomenon in Arabidopsis thaliana. Plant Mol. Biol. 81: 221-233
Stoll, B., Stoll, K., Steinhilber, J., Jonietz, C. and Binder, S.
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(2013) RNA PROCESSING FACTOR 5 is required for efficient 5’ cleavage at a processing site conserved in RNAs of three different mitochondrial genes in Arabidopsis thaliana. Plant J. 74: 593-604
Hauler, A., Jonietz, C., Stoll, B., Stoll, K., Braun, H.-P. and Binder, S.
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(2014) RNA processing factor 7 and polynucleotide phosphorylase are necessary for processing and stability of nad2 mRNA in Arabidopsis mitochondria. RNA Biol. 11: 968-976
Stoll, B., Zendler, D. and Binder, S.
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(2015) In Arabidopsis thaliana two co-adapted cyto-nuclear systems correlate with distinct ccmC transcript sizes. Plant J. 81: 247-257
Stoll, K., Jonietz, C. and Binder, S.
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(2017) In Arabidopsis thaliana distinct alleles encoding mitochondrial RNA PROCESSING FACTOR 4 support the generation of additional 5′ termini of ccmB transcripts. Plant Mol. Biol. 93: 659-668
Stoll, K., Jonietz, C., Schleicher, S., Colas des Francs-Small, C., Small I. and Binder, S.
