Apolipoprotein B mRNA sequences 3'' of the editing site are necessary and sufficient for editing and editosome assembly.

Apolipoprotein B (apoB) mRNA is edited in rat liver and intestine through the direct conversion of cytidine to uridine at nucleotide 6666. Recently, we have proposed the 'Mooring Sequence' model, in which editing complexes (editosomes) assemble on specific apoB mRNA flanking sequences to direct this site-specific editing event. To test this model, apoB mRNA deletion and translocation mutants were constructed and analyzed. Specific sequences 3' of the editing site were absolutely required for editing, while specific sequences and bulk RNA 5' of the editing site were required for efficient editing. Translocation of apoB 3' flanking sequences induced editing of an upstream cytidine, demonstrating that 3' sequences are necessary and sufficient to direct editing in vitro. 3' flanking sequences were also shown to be necessary and sufficient for editosome complex assembly. These data provide strong support for a 'Mooring Sequence' model in which 3' apoB flanking sequences direct editosome assembly and subsequent editing in vitro, while 5' flanking sequences enhance these functions.     

Pentatricopeptide repeat proteins constrain genome evolution in chloroplasts

Higher plants encode hundreds of pentatricopeptide repeat proteins (PPRs) that are involved in several types of RNA processing reactions. Most PPR genes are predicted to be targeted to chloroplasts or mitochondria, and many are known to affect organellar gene expression. In some cases, RNA binding has been directly demonstrated, and the sequences of the cis-elements are known. In this work, we demonstrate that RNA cis-elements recognized by PPRs are constrained in chloroplast genome evolution. Cis-elements for two PPR genes and several RNA editing sites were analyzed for sequence changes by pairwise nucleotide substitution frequency, pairwise indel frequency, and maximum likelihood (ML) phylogenetic distances. All three of these analyses demonstrated that sequences within the cis-element are highly conserved compared with surrounding sequences. In addition, we have compared sequences around chloroplast editing sites and homologous sequences in species that lack an editing site due to the presence of a genomic T. Cis-elements for RNA editing sites are highly conserved in angiosperms; by contrast, comparable sequences around a genomically encoded T exhibit higher rates of nucleotide substitution, higher frequencies of indels, and greater ML distances. The loss in requirement for editing to create the ndhD start codon has resulted in the conversion of the PPR gene responsible for editing that site to a pseudogene. We show that organellar dependence on nuclear-encoded PPR proteins for gene expression has constrained the evolution of cis-elements that are required at the level of RNA processing. Thus, the expansion of the PPR gene family in plants has had a dramatic effect on the evolution of plant organelle genomes.     

玉米不育系及其保持系线粒体atp6基因转录本编辑位点研究

以玉米同核异质细胞质雄性不育系T黄早四、C黄早四、S黄早四以及保持系N黄早四为材料, 比较研究了供试材料小孢子发育到单核期的线粒体atp6基因转录本保守区域的编辑位点。结果表明, DNA序列在T、C、S 3种胞质中完全一致, 与N胞质相比除在27、28核苷酸处不同外, 其余均一致, 而各胞质cDNA序列却不尽相同。DNA和cDNA序列比较显示: atp6基因转录本保守区域内, N、S胞质中均存在19个编辑位点, T胞质存在22个, C胞质存在20个, 它们相同的编辑位点有18个。大多数编辑位点都发生在密码子的第一、二位点上, 可改变氨基酸的种类。18个相同的编辑位点大都为完全编辑, 其中第1位点在各胞质中为部分编辑, 第19位点除在N胞质中为完全编辑外其余胞质都为部分编辑。而各胞质特有编辑位点均以部分编辑的形式出现。由此可见, 在玉米中atp6基因RNA编辑不仅具有序列特异性, 同时还受到胞质背景的影响。通过分析还可看出, 编辑的C残基前一个碱基多为嘧啶类碱基, 编码氨基酸Ser和Pro的密码子较其他类的密码子更易受到编辑, 且植物RNA的编辑有着改变蛋白质疏水性、增加物种间保守性的倾向。