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 evolution of chloroplast RNA editing

RNA editing alters the nucleotide sequence of an RNA molecule so that it deviates from the sequence of its DNA template. Different RNA-editing systems are found in the major eukaryotic lineages, and these systems are thought to have evolved independently. In this study, we provide a detailed analysis of data on C-to-U editing sites in land plant chloroplasts and propose a model for the evolution of RNA editing in land plants. First, our data suggest that the limited RNA-editing system of seed plants and the much more extensive systems found in hornworts and ferns are of monophyletic origin. Further, although some eukaryotic editing systems appear to have evolved to regulate gene expression, or at least are now involved in gene regulation, there is no evidence that RNA editing plays a role in gene regulation in land plant chloroplasts. Instead, our results suggest that land plant chloroplast C-to-U RNA editing originated as a mechanism to generate variation at the RNA level, which could complement variation at the DNA level. Under this model, many of the original sites, particularly in seed plants, have been subsequently lost due to mutation at the DNA level, and the function of extant sites is merely to conserve certain codons. This is the first comprehensive model for the evolution of the chloroplast RNA-editing system of land plants and may also be applicable to the evolution of RNA editing in plant mitochondria.     

银杏叶绿体ndhF RNA编辑现象分析及C290位编辑对胁迫处理的响应

RNA编辑是一种转录后修饰加工过程, 通过碱基的插入、缺失或替换可改变氨基酸的种类, 增加蛋白质的疏水性和同源蛋白在不同物种间的保守性。该文通过DNA与cDNA序列的比对, 分析了裸子植物银杏(Ginkgo biloba)叶绿体功能基因ndhF的编辑现象, 该基因共含有21个编辑位点, 且这21个位点均为部分编辑。生物信息学分析及与其它物种比对结果表明, ndhF C290位编辑可能会影响该蛋白的正确折叠。进一步使用单克隆酶切方法测定了不同胁迫处理对ndhF C290位编辑效率的影响, 结果表明该位点的编辑效率对温度和黑暗敏感。     

Methylation of ribosomal RNA genes inPetunia hybrida plants,callus cultures and regenerated shoots

The extent of methylation of the 18S–25S ribosomal RNA genes ofPetunia hybrida has been investigated using methylation-sensitive restriction endonucleases and Southern hybridisation. The genes in leaves, ovaries, petals, stigmas, anthers, and seed are methylated to a similar extent, but those in roots are hypomethylated. Genes in adventitious roots formed from stem cuttings are also hypomethylated. Genes in callus cultures initiated from leaf discs vary widely in the extent of methylation, but the pattern obtained is stable during culture. Callus cells containing hypomethylated genes can give rise to shoots in which the extent of methylation has reverted to that of the original leaf explant. These results demonstrate the striking plasticity of ribosomal RNA gene methylation inPetunia.     

TbRGG2 facilitates kinetoplastid RNA editing initiation and progression past intrinsic pause sites

TbRGG2 is an essential kinetoplastid RNA editing accessory factor that acts specifically on pan-edited RNAs. To understand the mechanism of TbRGG2 action, we undertook an in-depth analysis of edited RNA populations in TbRGG2 knockdown cells and an in vitro examination of the biochemical activities of the protein. We demonstrate that TbRGG2 down-regulation more severely impacts editing at the 5′ ends of pan-edited RNAs than at their 3′ ends. The initiation of editing is reduced to some extent in TbRGG2 knockdown cells. In addition, TbRGG2 plays a post-initiation role as editing becomes stalled in TbRGG2-depleted cells, resulting in an overall decrease in the 3′ to 5′ progression of editing. Detailed analyses of edited RNAs from wild-type and TbRGG2-depleted cells reveal that TbRGG2 facilitates progression of editing past intrinsic pause sites that often correspond to the 3′ ends of cognate guide RNAs (gRNAs). In addition, noncanonically edited junction regions are either absent or significantly shortened in TbRGG2-depleted cells, consistent with impaired gRNA transitions. Sequence analysis further suggests that TbRGG2 facilitates complete utilization of certain gRNAs. In vitro RNA annealing and in vivo RNA unwinding assays demonstrate that TbRGG2 can modulate RNA–RNA interactions. Collectively, these data are consistent with a model in which TbRGG2 facilitates initiation and 3′ to 5′ progression of editing through its ability to affect gRNA utilization, both during the transition between specific gRNAs and during usage of certain gRNAs.     

Identification of a pentatricopeptide repeat RNA editing factor in Physcomitrella patens chloroplasts

The moss Physcomitrella patens has two RNA editing sites in the chloroplasts. Here we identified a novel DYW-subclass pentatricopeptide repeat (PPR) protein, PpPPR_45, as a chloroplast RNA editing factor in P. patens. Knockdown of the PpPPR_45 gene reduced the extent of RNA editing at the chloroplast rps14-C2 site, whereas over-expression of PpPPR_45 increased the levels of RNA editing at both the rps14-C2 site and its neighboring C site. This indicates that the expression level of PpPPR_45 affects the extent of RNA editing at the two neighboring sites.