PPR蛋白参与RNA编辑机制的研究进展

RNA编辑是一种发生在转录后核苷酸特异位点的加工修饰现象,包括核苷酸的插入、删除和改变.高等植物中RNA编辑主要发生在线粒体与叶绿体中,具有重要的生物学功能,其机制仍在探索中.而PPR蛋白作为RNA编辑的反式作用因子,成为近几年来分子生物学的研究热点.该文就PPR蛋白、RNA编辑及PPR蛋白参与RNA编辑的机制等进行了综述.     

小麦叶绿体蛋白质编码基因RNA编辑位点的测定及与返白现象的关系

RNA编辑是一种转录后基因加工修饰现象,广泛存在于高等植物细胞器中。已有研究表明,RNA编辑与植物发生白化或者黄化有关。通过PCR、RT-PCR及测序的方法,对具有阶段性白化特性的小麦（Triticum aestivum）返白系FA85及其野生型矮变一号（Aibian 1）的叶绿体蛋白质编码基因RNA编辑位点进行了测定,在14个基因上发现了26个编辑位点。有5个编辑位点在2个株系之间存在编辑效率的差异,且这些差异的位点均位于编码叶绿体RNA聚合酶的基因上,其中3个位点编辑前后对应的蛋白质二级结构可能有差异。对2个株系叶绿体中PEP、NEP及PEP、NEP共同依赖基因转录水平的检测显示,除psbA和clpP外,其它基因在小麦返白系中的转录水平均有不同程度的下降。这种转录水平的显著下降及叶绿体RNA聚合酶基因上RNA编辑位点编辑效率的改变,可能与小麦返白系叶片的返白有关。     

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。     

白桦叶绿体RNA结合蛋白的蛋白质组学分析

在高等植物叶绿体中,RNA结合蛋白在转录后RNA处理、运输以及mRNA的稳定等方面发挥重要作用.本项研究使用多聚腺苷酸(polyA）吸附柱或单链DNA(ssDNA)吸附柱富集白桦叶绿体的polyA结合蛋白或RNA结合蛋白,并通过MALDI-TOF-MS以及ESI MS/MS进行鉴定,13个叶绿体蛋白质得到了鉴定.按照Swiss Prot数据库的注释,这些蛋白质的功能主要包括4个相关种类,分别为NAD结合蛋白、RNA结合蛋白、DNA结合蛋白和ATP结合蛋白.使用这些方法还鉴定出包括转录因子的4个高丰度蛋白.这些结果加深了对树木中叶绿体RNA结合蛋白的全面了解,可以将其应用于其他树木叶绿体中RNA 蛋白质的相互作用的研究.     

高等植物叶绿体表达重组蛋白研究进展

近年来,基因工程技术发展迅速,许多重组蛋白得以表达。其中利用植物生物反应器表达特异药物蛋白为人类一些重要疾病的预防和治疗提供了新途径。植物叶绿体遗传转化和表达系统成为目前植物生物反应器的研究热点。因结构和遗传上的特殊性,高等植物叶绿体在重组蛋白表达方面具有独特优势,外源基因表达量高、定点整合,而且叶绿体母系遗传特性保证了生物安全性。很多重要药用蛋白质在植物叶绿体中表达成功。烟草作为高等植物叶绿体转化模式植物,在疫苗抗原、抗体等药物蛋白和其他重要重组蛋白表达方面取得显著进展。高等植物叶绿体遗传转化也为叶绿体基因的表达和调控机制的研究提供新的技术和方法。文中从叶绿体遗传转化原理、载体构建、重组蛋白和重要药物蛋白在叶绿体中的表达以及重组蛋白表达对植物代谢和性状影响等多个角度,对高等植物叶绿体遗传转化体系研究的新进展进行了综述,以期为叶绿体表达平台的开发和重要药用蛋白质的表达提供新思路。     

油菜叶绿体4.5S rRNA与高梁ctDNA杂交及作为探针的应用

提取纯化了油菜叶绿体核糖体4.5S RNA(4.5S rRNA)并在其5’端标记 ³²P,作为探针与高梁叶绿体DNA(ctDNA)进行分子杂交。结果证明油菜4.5Sr RNA可作为公用探针,对一般植物,特别是高等植物进行分子杂交研究。杂交的结果得到两条带。     

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。     

台东苏铁中叶绿体功能基因RNA编辑的分析

台东苏铁是一种古老的裸子植物,有关苏铁的RNA编辑的研究很少。为此,我们分析了台东苏铁的RNA编辑位点和分布,为探究RNA编辑的功能和机制以及高等植物的起源和进化提供依据。本次以台东苏铁(Cycas taitungensis)为材料,采用异硫氰酸胍法提取台东苏铁总RNA。DNaseⅠ处理过的RNA逆转得到的c DNA进行PCR扩增条带并测序。通过对测序结果分析比对,初步确定在台东苏铁中的ndh D2,Pet B,ndh B2存在C-U的编辑。研究表明,ndh基因有较高的编辑频率,而丝氨酸相比其他氨基酸有更高的编辑频率。结果显示,ndh B2中有C-Y的部分编辑现象,ndh A2存在沉默编辑现象。     

RNA编辑修复藻苔(Takakia lepidozioides)叶绿体accD基因中的突变

藻苔(Takakia lepidozioides)叶绿体基因组中存在着非常高的RNA编辑频率,这种现象至今没有得到很好的解释。本研究采用比较基因组学方法,对小立碗藓(Physcomitrella patens)、青苔(Syntrichia ruralis)和藻苔3种苔藓植物中accD基因的DNA序列和基因编码的蛋白氨基酸序列进行比较分析。研究发现,经过RNA编辑,藻苔的基因产物获得了与其同源基因相同的氨基酸序列。藻苔中高频发生的RNA编辑是对基因组中DNA突变的一种修复,提高了藻苔对强辐射环境的生存适应能力。     

Site-selective inhibition of plastid RNA editing by heat shock and antibiotics: a role for plastid translation in RNA editing.

RNA editing in higher plant plastids changes single cytidine residues to uridine through an unknown mechanism. In order to investigate the relation of editing to physiological processes and to other steps in plastid gene expression, we have tested the sensitivity of chloroplast RNA editing to heat shock and antibiotics. We show that heat shock conditions as well as treatment of plants with prokaryotic translational inhibitors can inhibit plastid RNA editing. Surprisingly, this inhibitory effect is confined to a limited number of plastid editing sites suggesting that some site-specific factor(s) but none of the general components of the plastid RNA editing machinery are compromised. Contrary to previous expectations, our results provide evidence for a role of plastid translation in RNA editing.     

RNA editing in an untranslated region of the Ginkgo chloroplast genome.

mRNAs in plant cell organelles can be subject to RNA editing, an RNA processing step altering the identity of single nucleotide residues. In higher plant chloroplasts, editing proceeds by C-to-U conversions at highly specific sites. All known plastid RNA editing sites are located in protein-coding regions and, typically, change the coding properties of the mRNA. To gain more insight into the evolution of editing, we have determined the molecular structure and RNA editing pattern of the psbE operon of the primitive seed plant Ginkgo biloba. We report here the identification of altogether four sites of C-to-U editing, two of which are unique to Ginkgo and have not been found in other species. Surprisingly, one of the sites is located in an intercistronic spacer, thus being the first chloroplast editing site detected outside a protein-coding region. This indicates that the plastid editing machinery can operate also in untranslated regions and without having apparent functional consequences.     

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.     

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.