SILAC proteomics for quantitation of protein isoforms from alternative splicing in Arabidopsis seedlings

遗传变异是生物多样性和表型变异的重要基础。植物生长和生产力以及植物如何响应外部刺激如病原体/害虫或胁迫条件取决于植物物种的基因含量和基因表达的调节。基因在许多不同的水平上受到调控。一个重要的水平是基因的开启或关闭或向上或向下-称为转录控制。第二个层次发生在基因转录或复制成RNA后-称为转录后控制。转录后调控机制有很多种，选择性剪接（AS）是其中最重要的一种。选择性剪接是基因转录物的不同部分以不同的组合连接以从基因产生多于一个信使RNA（mRNA）的地方。所得到的mRNA可以被翻译成具有不同功能的蛋白质，或者可以被靶向降解。因此，AS增加了生物体的蛋白质组复杂性，并可以调节mRNA水平。选择性剪接影响植物发育、生存力、对外界条件的适应性、代谢和生理的许多方面，最近的估计表明植物中至少60%的含内含子基因经历AS。越来越多的例子被描述为AS调节表达或功能蛋白质多样性。随着我们对这一重要调控水平的全面了解的增加，有必要能够在蛋白质水平上研究AS的影响和动态变化。在鉴定和定量由AS产生的不同蛋白质同种型衍生的肽方面存在特别的挑战在各种基于质谱（MS）的定量蛋白质组学方法中，SILAC对于该特定应用具有许多优点：常规检测成千上万的蛋白质，通常相当大部分的蛋白质具有多个肽，蛋白质的定量相对简单，并且可以获得良好的MS分析软件。此外，SILAC能够定量低丰度肽和磷酸肽，并且已经开发了用于分析人细胞中蛋白质同种型的分析方法。SILAC在植物（细胞培养）中的使用非常有限，因为植物是自养的，用含稳定同位素的氨基酸标记效率低下。我们已经开发了一种方法来获得高水平的SILAC标记（>90%），更重要的是，在拟南芥幼苗（而不是细胞培养物）。这使得这项前沿技术首次适用于整个植物系统，并意味着它可以应用于植物生物学的广泛领域，从发育到对生物和非生物胁迫的反应，在本研究中，我们将使用SILAC技术的新改进，使其能够应用于植物幼苗，以研究选择性剪接在蛋白质水平上。我们将分析三组基因系，其中剪接因子或NMD因子过度表达或突变，并且我们从转录分析中得知许多基因的AS显著改变。这些细胞系将最大化鉴定同种型特异性肽的机会。目前关于植物中蛋白质异构体变体的数据很少，利用SILAC和使用特定的遗传系将使我们能够更好地了解AS在蛋白质水平上的影响。 最后，我们希望我们的植物SILAC系统能够更广泛地应用于其他非模式植物物种，并提供一种新的方法来研究翻译后修饰，蛋白质周转，microRNA对蛋白质水平的影响和蛋白质相互作用网络的生化问题。

Genetic variation is an important basis for biodiversity and phenotypic variation. Plant growth and productivity and how plants respond to external stimuli such as pathogens/pests or stress conditions depend on the gene content of the plant species and the regulation of expression of the genes. Genes are regulated at many different levels. One important level is where genes are turned on or off or up or down - called transcriptional control. A second level occurs after the gene is transcribed or copied into RNA - called post-transcriptional control. There are many different mechanisms of post-transcriptional control and alternative splicing (AS) is one of the most important. Alternative splicing is where different portions of a gene transcript are joined in different combinations to generate more than one messenger RNA (mRNA) from a gene. The resultant mRNAs can be translated into proteins with different functions or can be targeted for degradation. Thus, AS increases the proteome complexity of an organism and can regulate mRNA levels. Alternative splicing affects many aspects of plant development, viability, adaptability to external conditions, metabolism and physiology and the most recent estimate suggests that at least 60% of intron-containing genes in plants undergo AS. More and more examples are being described where AS regulates expression or functional protein diversity. As our overall knowledge of this important regulatory level increases, it becomes necessary to be able to investigate the impact and dynamic changes of AS at the protein level. There are particular challenges in identifying and quantifying peptides deriving from different protein isoforms generated by AS (e.g. the abundance of peptides that distinguish specific isoforms from peptides common to isoforms)Of various mass spectrometry (MS)-based quantitative proteomics methods, SILAC has a number of advantages for this specific application: many thousands of proteins are routinely detected, usually a significant fraction of proteins have multiple peptides, quantitation of proteins is relatively straightforward and good MS analysis software is available. In addition, SILAC,is able to quantify low abundance peptides and phosphopeptides, and analysis methods have been developed for analysis of protein isoforms in human cells. SILAC has had very limited use in plants (cell culture) due to inefficient labelling with stable-isotope-containing amino acids as plants are autotrophic. We have developed a method to obtain high levels of SILAC labelling (>90%) and, more significantly, in Arabidopsis seedlings (as opposed to cell cultures). This makes this frontline technology amenable to whole plant systems for the very first time and means that it can be applied to a wide range of areas of plant biology from development to responses to biotic and abiotic stresses, and facilitates the comparison of mutants at the protein level.In this proposal we will use this new modification of SILAC technology which allows it to be applied to plant seedlings to investigate alternative splicing at the protein level. We will analyse three sets of genetic lines where splicing factors or NMD factors are over-expressed or mutated and where we know from transcript analysis that the AS of many genes is significantly altered. These lines will maximise the opportunity for identifying isoform-specific peptides. There is currently very little data on protein isoform variants in plants and exploiting SILAC and using specific genetic lines will allow us to better understand the impact of AS at the protein level. Finally, we expect our plant SILAC system to be applied more widely to other, non-model plant species and to provide a new method to investigate biochemical questions of post-translational modification, protein turnover, microRNA effects on protein levels, and protein interaction networks.