RNA editing and DYW-type PPR proteins in the moss Physcomitrella patens
Zusammenfassung der Projektergebnisse
Obviously, all further refinements of the PPR-RNA binding code ultimately allowing to make reliable bioinformatic predictions for RNA targets of any newly identified PPR array sequence or, vice versa, to select the best fitting candidate PPR protein for a known RNA target sequence are highly welcome. In the future this could ultimately result in the combinatorial use of PPRs to design and engineer PPR proteins de novo to target specific RNA sequences. Physcomitrella patens wild-type as well as KO mutants affected specifically in mitochondrial RNA editing will be good test systems for changes introduced to the PPR arrays of DYW-type proteins targeted to mitochondria. Especially those Physcomitrella KO lines where a DYW-PPR knockout has not resulted in a recognizable impairment of vitality, such as our KO lines PPR_78 affecting editing sites cox1eU755SL and rps14eU137SL and PPR_79 affecting editing site nad5eU598RC will allow the testing of multiple PPR array variants in vivo. The engineered de novo targeting (e.g. of silent codon positions) of any of the numerous cytidines not affected by editing in the wild-type organelle transcriptome would be an obvious next step. Notably, Physcomitrella will remain an important alternative to angiosperm model systems like Arabidopsis thaliana for such purposes. Apparently, the RNA editing machinery is somewhat more complex in flowering plants with “E-type” PPR proteins lacking DYW domains and general editing factors called MORFs or RIPs, respectively, contributing to protein-protein interactions complicating the picture. In the absence of genes encoding such proteins in the Physcomitrella patens genome, its organelle RNA editing may reflect an evolutionary ancient, state with a 1:1 interaction of DYW-type PPR proteins and their cognate RNA targets. Likewise, protists such as Malawimonas and Naegleria may have retained an evolutionary ancestral, “pure” state with a 1:1 interaction of DYW-type PPR proteins and their RNA target sites in the mitochondria. The compact and size-reduced, but gene-rich, mitochondrial genomes of the protists and the apparently low numbers of DYW-type proteins and mitochondrial RNA editing sites (possibly also in rotifers) will be interesting additional data sets to improve our understanding of the PPR-RNA binding code. Even if not (yet) amenable to genetic manipulation these systems offer bioinformatic test cases for improving the PPR-RNA binding code since assignment predictions for factors and their RNA targets can be tested against the respective organelle genome sequences. Nevertheless, understanding how the apparently more complex RNA editing machinery in angiosperms ultimately selects candidate RNA targets over others is equally desirable. Given the moderate number of only ~30 chloroplast RNA editing sites in flowering plants, the chloroplast of the prime molecular model plant Arabidopsis thaliana will likely soon be second to the Physcomitrella mitochondrion as the next plant organelle transcriptome with a completed assignment of specificity factors to RNA editing sites. Given the ever increasing number of flowering plant genome and transcriptome sequence data being produced, which will reveal thousands of new PPR protein loci, insights on the corresponding organelle editomes will continue to be interesting. Accordingly, we will try to keep the PREPACT editome reference database updated and will aim to include in the annotation of editing sites the respective RNA editing protein factors identified and functionally characterized in model organisms. This should offer a helpful feature to understand the gain and loss of RNA editing sites and their respective specificity factors in the course of plant evolution. The organellar RNA editomes and the nuclear PPR gene families are a prominent example for co-evolution of two genetic systems within a cell that is not understood at present. Preliminary data recently obtained in our group show that in contrast to model angiosperms such as Arabidopsis or rice, early branching flowering plant and certain gymnosperm lineages contain more than 100 sites of RNA editing in their chloroplasts. Like in angiosperms, all of the gymnosperm RNA editing events are of the C-to-U type whereas the reverse U-to-C editing process is abundant in hornworts and may even surpass C-to-U editing in frequency in certain fern taxa. An improved understanding of the PPR-RNA binding code may help to identify a first candidate editing factor for a U-to-C editing event in those taxa. Notably, it must be remembered that despite ever increasing circumstantial evidence for a cytidine deaminase activity of the DYW domain, any direct biochemical proof for this is at present still lacking and the possibility of a transaminase activity that could catalyze the pyrimidine exchange editing in both directions using an as yet unidentified transamination partner should be kept in mind. Finally, with the discoveries of sporadic occurrences of DYW-type PPR proteins outside of land plants, the puzzling evolutionary origin of RNA editing and the reasons for its persisting existence in land plants are no less mysterious than before. At present, horizontal gene transfers (HGTs) of DYW-PPR genes would explain their phylogenetic distribution more parsimoniously than numerous independent losses in many groups of eukaryotes. Consequently, this would suggest that such events of HGT with functional DYW-type PPR genes persisting in the genome of the new hosts would subsequently allow for RNA editing to become established as a correcting mechanisms for T-to-C mutations in the mitochondrial genomes. Careful inspection of future genome data will show whether this idea can be further corroborated.
Projektbezogene Publikationen (Auswahl)
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(2009) RNA editing: only eleven sites are present in the Physcomitrella patens mitochondrial transcriptome and a universal nomenclature proposal. Molecular Genetics and Genomics 281, 473-481
Rüdinger M, Funk HT, Rensing SA, Maier UG, Knoop V
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(2010) DYW-type PPR proteins in a heterolobosean protist: Plant RNA editing factors involved in an ancient horizontal gene transfer? FEBS Letters 584, 4287-4291
Knoop V, Rüdinger M
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(2011) Assigning DYW-type PPR proteins to RNA editing sites in the funariid mosses Physcomitrella patens and Funaria hygrometrica. Plant Journal 67, 370-380
Rüdinger M, Szövényi P, Rensing SA, Knoop V
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(2011) Plant-type mitochondrial RNA editing in the protist Naegleria gruberi. RNA 17, 2058-2062
Rüdinger M, Fritz-Laylin L, Polsakiewicz M, Knoop V
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(2012) Nuclear DYW-type PPR gene families diversify with increasing RNA editing frequencies in liverwort and moss mitochondria. Journal of Molecular Evolution 74, 37-51
Rüdinger M, Volkmar U, Lenz H, Groth-Malonek M, Knoop V
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(2013) A DYW-protein knockout in Physcomitrella affects two closely spaced mitochondrial editing sites and causes a severe developmental phenotype. Plant Journal 76, 420-432
Schallenberg-Rüdinger M, Kindgren P, Zehrmann A, Small I, Knoop V
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(2013) A survey of PPR proteins identifies DYW domains like those of land plant RNA editing factors in diverse eukaryotes. RNA Biol 10, 1343-1350
Schallenberg-Rüdinger M, Lenz H, Polsakiewicz M, Gott JM, Knoop V
