The DYW-E-PPR protein MEF14 is required for RNA editing at site matR-1895 in mitochondria of Arabidopsis thaliana

We here identify the PPR protein MEF14 of the DYW subclass as a specific trans-factor required for C to U editing of site matR-1895 by genetic mapping of an EMS induced editing mutant in Arabidopsis thaliana. The wild type Col MEF14 gene complements mutant protoplasts. A T-DNA insertion in the MEF14 gene abolishes detectable editing at the matR-1895 site. Lack of RNA editing at the matR-1895 site does not alter the level of mature and precursor nad1 mRNA molecules. Such RNA editing mutants can be used to analyse the function of genes like this maturase related reading frame in plant mitochondria.     

Seven large variations in the extent of RNA editing in plant mitochondria between three ecotypes of Arabidopsis thaliana

Most RNA editing sites in flowering plant mitochondria are located in coding regions of mRNAs and are usually essential for correct gene expression. Although accordingly little variation should be tolerated, editing sites appear and disappear even between closely related flowering plant species. To investigate whether such editing site variations also occur within species, we analyzed 379 RNA editing sites in the three ecotypes Columbia, Landsberg erecta and C24 of Arabidopsis thaliana. While all editing sites as such are conserved, we identify seven RNA editing sites with 40-60% differences in effective editing between individual ecotypes. These quantitative variations show that the extent of RNA editing in plant mitochondria is very flexible and can change even more rapidly than the evolution of species. The ecotype-specific variations of the RNA editing extent are Mendelian-inherited and can now be used to follow and identify the nuclear loci responsible for these RNA editing phenotypes.     

The DYW Subgroup PPR Protein MEF35 Targets RNA Editing Sites in the Mitochondrial rpl16, nad4 and cob mRNAs in Arabidopsis thaliana

RNA editing in plant mitochondria and plastids alters specific nucleotides from cytidine (C) to uridine (U) mostly in mRNAs. A number of PLS-class PPR proteins have been characterized as RNA recognition factors for specific RNA editing sites, all containing a C-terminal extension, the E domain, and some an additional DYW domain, named after the characteristic C-terminal amino acid triplet of this domain. Presently the recognition factors for more than 300 mitochondrial editing sites are still unidentified. In order to characterize these missing factors, the recently proposed computational prediction tool could be of use to assign target RNA editing sites to PPR proteins of yet unknown function. Using this target prediction approach we identified the nuclear gene MEF35 (Mitochondrial Editing Factor 35) to be required for RNA editing at three sites in mitochondria of Arabidopsis thaliana. The MEF35 protein contains eleven PPR repeats and E and DYW extensions at the C-terminus. Two T-DNA insertion mutants, one inserted just upstream and the other inside the reading frame encoding the DYW domain, show loss of editing at a site in each of the mRNAs for protein 16 in the large ribosomal subunit (site rpl16-209), for cytochrome b (cob-286) and for subunit 4 of complex I (nad4-1373), respectively. Editing is restored upon introduction of the wild type MEF35 gene in the reading frame mutant. The MEF35 protein interacts in Y2H assays with the mitochondrial MORF1 and MORF8 proteins, mutation of the latter also influences editing at two of the three MEF35 target sites. Homozygous mutant plants develop indistinguishably from wild type plants, although the RPL16 and COB/CYTB proteins are essential and the amino acids encoded after the editing events are conserved in most plant species. These results demonstrate the feasibility of the computational target prediction to screen for target RNA editing sites of E domain containing PLS-class PPR proteins.     

The E-class PPR protein MEF3 of Arabidopsis thaliana can also function in mitochondrial RNA editing with an additional DYW domain

In plants, RNA editing is observed in mitochondria and plastids, changing selected C nucleotides into Us in both organelles. We here identify the PPR (pentatricopeptide repeat) protein MEF3 (mitochondrial editing factor 3) of the E domain PPR subclass by genetic mapping of a variation between ecotypes Columbia (Col) and Landsberg erecta (Ler) in Arabidopsis thaliana to be required for a specific RNA editing event in mitochondria. The Ler variant of MEF3 differs from Col in two amino acids in repeats 9 and 10, which reduce RNA editing levels at site atp4-89 to about 50% in Ler. In a T-DNA insertion line, editing at this site is completely lost. In Vitis vinifera the gene most similar to MEF3 continues into a DYW extension beyond the common E domain. Complementation assays with various combinations of PPR and E domains from the vine and A. thaliana proteins show that the vine E region can substitute for the A. thaliana E region with or without the DYW domain. These findings suggest that the additional DYW domain does not disturb the MEF3 protein function in mitochondrial RNA editing in A. thaliana.     

Recent stable insertion of mitochondrial DNA into an Arabidopsis polyubiquitin gene by nonhomologous recombination.

Sequence analysis of a newly identified polyubiquitin gene (UBQ13) from the Columbia ecotype of Arabidopsis thaliana revealed that the gene contained a 3.9-kb insertion in the coding region. All subclones of the 3.9-kb insert hybridized to isolated mitochondrial DNA. The insert was found to consist of at least two, possibly three, distinct DNA segments from the mitochondrial genome. A 590-bp region of the insert is nearly identical to the Arabidopsis mitochondrial nad1 gene. UBQ13 restriction fragments in total cellular DNA from ecotypes Ler, No-0, Be-0, WS, and RLD were identified and, with the exception of Be-0, their sizes were equivalent to that predicted from the corresponding ecotype Columbia UBQ13 restriction fragment without the mitochondrial insert. Isolation by polymerase chain reaction and sequence determination of UBQ13 sequences from the other ecotypes showed that all lacked the mitochondrial insert. All ecotypes examined, except Columbia, contain intact open reading frames in the region of the insert, including four ubiquitin codons which Columbia lacks. This indicates that the mitochondrial DNA in UBQ13 in ecotype Columbia is the result of an integration event that occurred after speciation of Arabidopsis rather than a deletion event that occurred in all ecotypes except Columbia. This stable movement of mitochondrial DNA to the nucleus is so recent that there are few nucleotide changes subsequent to the transfer event. This allows for precise analysis of the sequences involved and elucidation of the possible mechanism. The presence of intron sequences in the transferred nucleic acid indicates that DNA was the transfer intermediate. The lack of sequence identity between the integrating sequence and the target site, represented by the other Arabidopsis ecotypes, suggests that integration occurred via nonhomologus recombination. This nuclear/organellar gene transfer event is strikingly similar to the experimentally accessible process of nuclear integration of introduced heterologous DNA.     

Mitochondrial gene transfer in pieces: fission of the ribosomal protein gene rpl2 and partial or complete gene transfer to the nucleus.

Mitochondrial genes are usually conserved in size in angiosperms. A notable exception is the rpl2 gene, which is considerably shorter in the eudicot Arabidopsis than in the monocot rice. Here, we show that a severely truncated mitochondrial rpl2 gene (termed 5' rpl2) was created by the formation of a premature stop codon early in eudicot evolution. This 5' rpl2 gene was subsequently lost many times from the mitochondrial DNAs of 179 core eudicots surveyed by Southern hybridization. The sequence corresponding to the 3' end of rice rpl2 (termed 3' rpl2) has been lost much more pervasively among the mitochondrial DNAs of core eudicots than has 5' rpl2. Furthermore, where still present in these mitochondrial genomes, 3' rpl2 always appears to be a pseudogene, and there is no evidence that 3' rpl2 was ever a functional mitochondrial gene. An intact and expressed 3' rpl2 gene was discovered in the nucleus of five diverse eudicots (tomato, cotton, Arabidopsis, soybean, and Medicago). In the first three of these species, 5' rpl2 is still present in the mitochondrion, unlike the two legumes, where both parts of rpl2 are present in the nucleus as separate genes. The full-length rpl2 gene has been transferred intact to the nucleus in maize. We propose that the 3' end of rpl2 was functionally transferred to the nucleus early in eudicot evolution, and that this event then permitted the nonsense mutation that gave rise to the mitochondrial 5' rpl2 gene. Once 5' rpl2 was established as a stand-alone mitochondrial gene, it was then lost, and was probably transferred to the nucleus many times. This complex history of gene fission and gene transfer has created four distinct types of rpl2 structures or compartmentalizations in angiosperms: (1) intact rpl2 gene in the mitochondrion, (2) intact gene in the nucleus, (3) split gene, 5' in the mitochondrion and 3' in the nucleus, and (4) split gene, both parts in the nucleus.     

Divergent intron conservation in the mitochondrial nad2 gene: signatures for the three bryophyte classes (mosses,liverworts, and hornworts) and the lycophytes

The slow-evolving mitochondrial DNAs of plants have potentially conserved information on the phylogenetic branching of the earliest land plants. We present the nad2 gene structures in hornworts and liverworts and in the presumptive earliest-branching vascular land plant clade, the Lycopodiopsida. Taken together with the recently obtained nad2 data for mosses, each class of bryophytes presents another pattern of angiosperm-type introns conserved in nad2: intron nad2i1 in mosses; intron nad2i3 in liverworts; and both introns, nad2i3 and nad2i4, in hornworts. The lycopods Isoetes and Lycopodium show diverging intron conservation and feature a unique novel intron, termed nad2i3b. Hence, mitochondrial introns in general are positionally stable in the bryophytes and provide significant intraclade phylogenetic information, but the nad2 introns, in particular, cannot resolve the interclade relationships of the bryophyte classes and to the tracheophytes. The necessity for RNA editing to reconstitute conserved codon entities in nad2 is obvious for all clades except the marchantiid liverworts. Finally, we find that particularly small group II introns appear as a general feature of the Isoetes chondriome. Plant mitochondrial peculiarities such as RNA editing frequency, U-to-C type of RNA editing, and small group II introns appear to be genus-specific rather than gene-specific features.     

陆地棉线粒体nad6基因mRNA无常规终止密码子

植物线粒体nad6基因编码NADH（还原型辅酶Ⅰ）脱氢酶第6亚基,前期研究发现,该基因可能与棉花细胞质雄性不育相关,但该基因的转录情况尚不清楚.本研究利用PCR测序、Southern印迹方法发现,陆地棉线粒体基因组（mtDNA）中nad6基因长621 bp,且为单拷贝. RT-PCR及环化RT-PCR分析发现,其mRNA在终止密码子前-14或-15 nt处提前终止|虽然该基因mRNA编码区存在12处RNA编辑（C-U）位点,但并未产生新的替代的终止密码子|基因mRNAs尾端poly（A）处含有0、1、2或4个“A”,也并未与前方相邻碱基凑成新的终止密码子,即：该基因mRNA无常规终止密码子（UGA,UAG,UAA）.本研究结果提示,棉花线粒体nad6基因mRNA很可能有其它的终止密码子.针对这种情况对植物线粒体如何翻译无常规终止密码子的mRNA进行了讨论.