Abstract
RNA editing in flowering plant mitochondria post-transcriptionally alters several hundred nucleotides from C to U, mostly in mRNAs. Several factors required for specific RNA-editing events in plant mitochondria and plastids have been identified, all of them PPR proteins of the PLS subclass with a C-terminal E-domain and about half also with an additional DYW domain. Based on this information, we here probe the connection between E-PPR proteins and RNA editing in plant mitochondria. We initiated a reverse genetics screen of T-DNA insertion lines in Arabidopsis thaliana and investigated 58 of the 150 E-PPR-coding genes for a function in RNA editing. Six genes were identified to be involved in mitochondrial RNA editing at specific sites. Homozygous mutants of the five genes MEF18-MEF22 display no gross disturbance in their growth or development patterns, suggesting that the editing sites affected are not crucial at least in the greenhouse. These results show that a considerable percentage of the E-PPR proteins are involved in the functional processing of site-specific RNA editing in plant mitochondria.
Highlights
Specific sequence contexts in the pre-mRNA yield the first part of an answer; such cis-elements are required to identify a bona fide editing site as in vivo, in organelle, and in vitro analyses of several mitochondrial RNA-editing sites have shown [5,6,7,8,9,10,11]
The second part of the answer seems to be provided by the identification of nuclear encoded specificity factors in Arabidopsis thaliana such as MEF1, MEF9, and MEF11 (mitochondrial-editing factor (MEF)2), which are required intact for correct editing of specific different sites [12,13,14,15]
We addressed the first question and initiated an analysis to test if other proteins similar to MEF1, MEF9, and MEF11 are involved in RNA editing in plant mitochondria
Summary
Plant Material and Preparation of Nucleic Acids—A. thaliana seeds for the Columbia (Col) ecotype were kind gifts from J. The T-DNA insertion lines of A. thaliana were obtained from TAIR resources. Growth of the A. thaliana plants and preparation of DNA or RNA from leaves were as described [39]. Seeds were sown as obtained and used for the initial screening in material from pools of eight plants. Identified mutants were selfed, and the T-DNA insertion sites and homozygocity of individuals were verified by PCR and sequence analysis. SNaPshot Assays and Mutant Analysis—The 58 T-DNA insertion lines in E-PPR-proteins and the 3 in PLS-PPRs were screened by multiplexed single base extension [38] for plants with altered RNA editing at specific sites. In the identified individual plants, the compromised RNA-editing phenotype was verified by cDNA sequence analysis for the status of the respective investigated editing site. RNA-editing levels were estimated by the relative areas of the respective nucleotide peaks in the sequence analyses
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