Proteomic LC-MS analysis of Arabidopsis cytosolic ribosomes: Identification of ribosomal protein paralogs and re-annotation of the ribosomal protein genes
Proteomic LC-MS analysis of Arabidopsis cytosolic ribosomes: Identification of ribosomal protein paralogs and re-annotation of the ribosomal protein genes
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DOI：
10.1016/j.jprot.2015.07.004
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发表时间：
2015-10-14
影响因子：
3.3
通讯作者：
Hanson, Johannes
中科院分区：
文献类型：
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作者：
Hummel, Maureen;Dobrenel, Thomas;Hanson, Johannes
拟南芥胞质核糖体是由81个不同的核糖体蛋白(r-蛋白)、4个核糖体RNA(RRNA)和大量相关(非核糖体)蛋白组成的大型复合体。在植物中，胞质核糖体的r-蛋白由两到七个不同表达和相似的基因编码，形成一个r-蛋白家族，基因家族成员r-蛋白编码序列的差异是核糖体之间差异的来源。我们对主动翻译的胞质核糖体进行了蛋白质组学研究，这些核糖体是通过免疫提纯和经典的蔗糖缓冲离心法从不同发育阶段的植物中纯化出来的。使用数据独立采集的一维和二维LC MSE以及常规数据依赖的MS/MS程序。该方法提供了基于蛋白质型肽的165个高覆盖率的r-蛋白同源基因的详细鉴定。检测到的r-蛋白是基因组编码的242个胞质r-蛋白基因中大部分(68%)的产物。共鉴定出70个不同的r-蛋白。基于这些结果和来自DNA微阵列和核糖体足迹图谱研究的信息，我们提出了对拟南芥r-蛋白和基因的重新注释。这一细胞质r蛋白蛋白质组概要将作为未来研究植物核糖体动态结构和功能的模板。生物学意义：翻译是活细胞中最需要能量的过程之一，因此受到仔细的调控。翻译活动与生长控制和生长调控机制密切相关。最近建立的翻译图谱技术，包括与多聚体相关的mRNAs图谱和mRNAs上核糖体足迹的图谱，揭示了基因表达往往通过基因转录本的差异翻译来微调。核糖体是这些重要过程的中枢，由近80种不同的蛋白质(取决于物种)和四个大的RNA组成，组装成两个高度保守的亚基。在植物中，在酵母中，r-蛋白由多个活跃转录的基因编码。由于r-蛋白基因同源序列通常不编码相同的蛋白质，并且受生长条件和发育的调节，体内核糖体的蛋白质含量是不同的，这种异质性的调节和生理意义尚不清楚。在这里，提出了一种结合蛋白质组学和高级mRNA表达数据的对拟南芥200多个r-蛋白基因的改进注释。这项对拟南芥核糖体的蛋白质组学研究和重新注释为未来植物翻译调控的研究奠定了基础。(C)2015爱思唯尔B.V.保留所有权利。
Arabidopsis thaliana cytosolic ribosomes are large complexes containing eighty-one distinct ribosomal proteins (r-proteins), four ribosomal RNAs (rRNA) and a plethora of associated (non-ribosomal) proteins. In plants, r-proteins of cytosolic ribosomes are each encoded by two to seven different expressed and similar genes, forming an r-protein family.Distinctions in the r-protein coding sequences of gene family members are a source of variation between ribosomes. We performed proteomic investigation of actively translating cytosolic ribosomes purified using both immunopurification and a classic sucrose cushion centrifugation-based protocol from plants of different developmental stages. Both 1D and 2D LC MSE with data-independent acquisition as well as conventional data-dependent MS/MS procedures were applied. This approach provided detailed identification of 165 r-protein paralogs with high coverage based on proteotypic peptides. The detected r-proteins were the products of the majority (68%) of the 242 cytosolic r-protein genes encoded by the genome. A total of 70 distinct r-proteins were identified. Based on these results and information from DNA microarray and ribosome footprint profiling studies a re-annotation of Arabidopsis r-proteins and genes is proposed. This compendium of the cytosolic r-protein proteome will serve as a template for future investigations on the dynamic structure and function of plant ribosomes.Biological significance: Translation is one of the most energy demanding processes in a living cell and is therefore carefully regulated. Translational activity is tightly linked to growth control and growth regulating mechanism. Recently established translational profiling technologies, including the profiling of mRNAs associated with polysomes and the mapping of ribosome footprints on mRNAs, have revealed that the expression of gene expression is often fine-tuned by differential translation of gene transcripts. The eukaryotic ribosome, the hub of these important processes, consists of close to eighty different proteins (depending on species) and four large RNAs assembled into two highly conserved subunits.In plants and to lesser extent in yeast the r-proteins are encoded by more than one actively transcribed gene. As r-protein gene paralogs frequently do not encode identical proteins and are regulated by growth conditions and development, in vivo ribosomes are heterogeneous in their protein content The regulatory and physiological importance of this heterogeneity is unknown. Here, an improved annotation of the more than two hundred r-protein genes of Arabidopsis is presented that combines proteomic and advanced mRNA expression data. This proteomic investigation and re-annotation of Arabidopsis ribosomes establish a base for future investigations of translational control in plants. (C) 2015 Elsevier B.V. All rights reserved.