植物细胞器RNA编辑因子的功能及其作用机制 *

在植物线粒体和叶绿体转录本上,数百个胞嘧啶(C)位点经脱氨基反应变为尿嘧啶(U),这是一种在转录本水平上对遗传信息进行修饰或调控的机制.在植物细胞器中,RNA编辑过程需要不同家族的RNA编辑因子相互作用组装成复杂的编辑复合体,特异地识别编辑位点进行编辑.最初的研究发现,植物RNA编辑受到高特异性五环肽重复(pentatricopeptide repeat, PPR)蛋白的调控,目前在植物中发现400多种PPR家族蛋白,编辑作用复杂.之后对RNA编辑因子互作蛋白/多细胞器RNA编辑因子(RNA editing factor interacting proteins /multiple organellar RNA editing factors,RIP/MORF),细胞器RNA识别基序(organelle RNA recognition motif,ORRM),细胞器锌指蛋白(organelle zinc-finger,OZ)等的研究表明,这些非PPR蛋白组分可以与PPR蛋白形成编辑复合体,共同参与编辑,且RNA编辑复合体具有多样性.RNA编辑因子的缺失会引起植物的生长发育受阻,果实成熟延迟等,对RNA编辑因子的研究显得尤为重要.对植物中RNA编辑因子的功能及其作用机制研究进展进行综述,旨在为后续RNA编辑的研究提供一定的参考.     

植物细胞器RNA编辑因子的功能及其作用机制

在植物线粒体和叶绿体转录本上,数百个胞嘧啶(C)位点经脱氨基反应变为尿嘧啶(U),这是一种在转录本水平上对遗传信息进行修饰或调控的机制。在植物细胞器中,RNA编辑过程需要不同家族的RNA编辑因子相互作用组装成复杂的编辑复合体,特异地识别编辑位点进行编辑。最初的研究发现,植物RNA编辑受到高特异性五环肽重复(pentatricopeptide repeat,PPR)蛋白的调控,目前在植物中发现400多种PPR家族蛋白,编辑作用复杂。之后对RNA编辑因子互作蛋白/多细胞器RNA编辑因子(RNA editing factor interacting proteins/multiple organellar RNA editing factors,RIP/MORF)、细胞器RNA识别基序(organelle RNA recognition motif,ORRM)、细胞器锌指蛋白(organelle zinc-finger,OZ)等的研究表明,这些非PPR蛋白组分可以与PPR蛋白形成编辑复合体,共同参与编辑,且RNA编辑复合体具有多样性。RNA编辑因子的缺失会引起植物的生长发育受阻、果实成熟延迟等,对RNA编辑因子的研究显得尤为重要。对植物中RNA编辑因子的功能及其作用机制研究进展进行综述,旨在为后续RNA编辑的研究提供一定的参考。     

PPR蛋白调控植物细胞器RNA加工的分子功能

35肽重复（pentatricopeptide repeat, PPR）蛋白是2000年发现的一类由多个重复单位串联而成的核编码蛋白质。PPR蛋白广泛存在于真核生物中,在陆生植物中尤为普遍。PPR蛋白大多定位于线粒体或叶绿体。多项研究表明,PPR蛋 白为序列特异性RNA结合蛋白质,在细胞器RNA编辑、剪接、稳定、切割及翻译等转录后加工过程发挥重要作用。PPR蛋白功能缺陷导致植物生长发育异常,甚至胚胎致死。本文主要就PPR蛋白功能及作用机制进行综述,并对尚待解决的问题及研究前景加以探讨,以期为PPR蛋白的深入研究提供思路。     

PPR蛋白影响植物生长发育的研究进展

植物生长发育是一个极其复杂的生理生化过程,受内外因素共同作用。PPR蛋白是核基因编码的具有重复PPR基序的蛋白,分布广泛,在高等植物中数量巨大。PPR蛋白的靶标一般是线粒体和叶绿体中转录的RNA前体,多数可与MORF互作,参与线粒体和叶绿体基因的RNA编辑。PPR蛋白缺失的突变体植株多数呈现异常表型,影响植物的正常生长发育。本文就近年来发现的PPR蛋白结构、分布,与RNA编辑的关系,及其对植物生长发育的影响进行了综述。     

PPR蛋白在植物生长发育中的作用

PPR蛋白在陆生植物中属于最大的蛋白家族之一,其成员种类和数量均十分庞大。PPR蛋白主要的功能是通过在多种细胞器中进行定位从而参与细胞核和细胞器中特异单链RNA的转录后修饰和编辑,在植物生长发育的多个阶段均发挥着重要的作用。多数PPR蛋白编码基因的突变体呈现异常的发育表型,如胚胎致死、发育迟缓及绿化延迟等。对近年来植物PPR蛋白的分类、定位、RNA修饰的机制及其对植物生长发育影响进行了综述,并展望了植物PPR发挥功能区域和参与的调控网络研究。     

PPR蛋白在水稻生长发育中的功能研究进展

水稻是我国最主要的粮食作物之一.水稻的安全生产越来越依赖分子生物学功能基础研究.PPR(Pentatricopeptide repeat)蛋白是水稻中较大的蛋白家族之一,研究证明PPR蛋白由核基因编码并参与细胞器前体RNA加工,如RNA起始翻译、RNA稳定、RNA剪接、RNA编辑,调控水稻叶绿体和线粒体等的发育,进而影...     

RNA编辑的研究进展

RNA编辑,即通过碱基的插入、删除和替换对RNA进行的转录后加工过程,这一表观遗传现象也被认为是在RNA水平上对遗传信息进行修复的一种修正机制.本文主要综述了目前植物中基于PPR基因家族等编辑复合体以及动物中关于CRISPR/Cas系统的两种RNA编辑系统,并介绍了RNA编辑在植物生长发育过程中的重要作用,并展望了RN...     

水稻线粒体coxII基因转录本的编辑位点研究

RNA编辑是指由RNA水平的核苷酸改变所引起的密码子发生变化的一种预定修饰,它的发现是近年来对分子生物学中心法则的重要补充.本文以红莲型(HL)水稻细胞质雄性不育系粤泰A,保持系粤泰B和杂种F1(不育系A与恢复系71068的杂交一代)为材料,首次研究了线粒体基因coxII转录本的编辑位点.coxII基因的转录本有15个编辑位点,其中有14个发生在密码子的第一和第二位点.这14个位点的编辑可改变氨基酸的种类,并导致所编码蛋白的疏水性以及所编码蛋白在氨基酸序列上的保守性增加.     

番茄PPR基因家族的鉴定与生物信息学分析

PPR(Pentatricopeptide repeats)基因家族在植物中广泛存在, 其在植物生长发育过程中至关重要。文章采用生物信息学方法, 利用Pfam已鉴定的PPR保守结构域序列检索番茄(Solanum lycopersicum L.)基因组计划注释的蛋白序列, 最终确定了番茄中可能存在的471个PPR编码基因; 根据拟南芥(Arabidopsis thaliana L.)中鉴定的各个结构域的特点对其进行了蛋白结构分析、分类和保守序列分析, 并对番茄PPR基因家族进行了系统进化树构建、染色体定位、亚细胞定位预测、表达和GO分析等。结果表明：番茄PPR基因家族分为P和PLS两个亚家族, 各占序列数目的一半, PLS亚家族又分为PLS、E、E+和DYW四类, 且在进化树中形成不同的分支; 各个结构域在植物中非常保守; PPR基因家族分布在番茄12条染色体上, 且多数无内含子结构; 大部分PPR蛋白具有线粒体或叶绿体定位序列, GO分析表明PPR蛋白参与RNA相关的生物学过程     

RNA编辑功能和RNA干扰

RNA编辑被认为是生命体一种新的基因加工与修饰现象,是指DNA转录成RNA后除RNA剪切外的其他加工过程,以核苷酸的删除、插入或替换等方式改变遗传信息,揭示生物进化过程中基因修饰和调控的另一个重要途径,是对中心法则的重要补充.而RNAi是一种由dsRNA介导的,在转录水平、转录后水平和翻译水平上阻断基因表达的基因调节途径.着重介绍 RNA编辑功能、RNA编辑与RNA干扰关系.     

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.     

The potential for manipulating RNA with pentatricopeptide repeat proteins

The pentatricopeptide repeat (PPR) protein family, which is particularly prevalent in plants, includes many sequence‐specific RNA‐binding proteins involved in all aspects of organelle RNA metabolism, including RNA stability, processing, editing and translation. PPR proteins consist of a tandem array of 2‐30 PPR motifs, each of which aligns to one nucleotide in the RNA target. The amino acid side chains at two or three specific positions in each motif confer nucleotide specificity in a predictable and programmable manner. Thus, PPR proteins appear to provide an extremely promising opportunity to create custom RNA‐binding proteins with tailored specificity. We summarize recent progress in understanding RNA recognition by PPR proteins, with a particular focus on potential applications of PPR‐based tools for manipulating RNA, and on the challenges that remain to be overcome before these tools may be routinely used by the scientific community.     

Improved Computational Target Site Prediction for Pentatricopeptide Repeat RNA Editing Factors

Pentatricopeptide repeat (PPR) proteins with an E domain have been identified as specific factors for C to U RNA editing in plant organelles. These PPR proteins bind to a unique sequence motif 5′ of their target editing sites. Recently, involvement of a combinatorial amino acid code in the P (normal length) and S type (short) PPR domains in sequence specific RNA binding was reported. PPR proteins involved in RNA editing, however, contain not only P and S motifs but also their long variants L (long) and L2 (long2) and the S2 (short2) motifs. We now find that inclusion of these motifs improves the prediction of RNA editing target sites. Previously overlooked RNA editing target sites are suggested from the PPR motif structures of known E-class PPR proteins and are experimentally verified. RNA editing target sites are assigned for the novel PPR protein MEF32 (mitochondrial editing factor 32) and are confirmed in the cDNA.     

Predictable Alteration of Sequence Recognition by RNA Editing Factors from Arabidopsis

RNA editing factors of the pentatricopeptide repeat (PPR) family show a very high degree of sequence specificity in the recognition of their target sites. A molecular basis for target recognition by editing factors has been proposed based on statistical correlations but has not been tested experimentally. To achieve this, we systematically mutated the pentatricopeptide motifs in the Arabidopsis thaliana RNA editing factor CLB19 to investigate their individual contribution to RNA recognition. We find that the motifs contributing significantly to the specificity of binding follow the previously proposed recognition rules, distinguishing primarily between purines and pyrimidines. Our results are consistent with proposals that each motif recognizes one nucleotide in the RNA target with the protein aligned parallel to the RNA and contiguous motifs aligned with contiguous nucleotides such that the final PPR motif aligns four nucleotides upstream of the edited cytidine. By altering S motifs in CLB19 and another editing factor, OTP82, and using the modified proteins to attempt to complement the respective mutants, we demonstrate that we can predictably alter the specificity of these factors in vivo.