Activation of an Endoribonuclease by Non-intein Protein Splicing

通过非内含肽蛋白质剪接激活核糖核酸内切酶

• 批准号: 1244106
• 负责人: David Stern
• 金额: $ 44.51万
• 依托单位: Boyce Thompson Institute Plant Research
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-05-01 至 2016-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1244106&HistoricalAwards=false
• 关键词: Activation; Endoribonuclease; Non; intein; Protein

The sequence of a protein can be predicted from that of the RNA which specifies it. In other words, there is usually a correspondence between the code embedded in an RNA molecule, and the protein resulting from the activity of that RNA as a template. A protein called RB47 fails to follow this paradigm and is the main focus of this project. Specifically, RB47 is synthesized according to its RNA template, but then an internal portion of the protein is removed, with the two flanking portions being combined into a spliced, smaller protein. Such protein splicing reactions are known in nature, but are normally spontaneous. In the case of RB47, specific cellular factors are required to splice it. The research is focused around the mechanism of this splicing, which has no obvious precedent. A second aspect of RB47 is that the initial version of the protein is enzymatically inert, but the spliced version harbors an RNA cleavage activity. Thus, protein splicing activates this enzyme. This hints that the splicing has a regulatory function as well. The project has three specific goals: first, to define the signals within the protein itself that are required for RNA cleavage activity; second, to discover the molecular apparatus that carries out the splicing reaction; and third, to use genetic experiments to discern the biological role of RB47. Although RB47 is a protein from the chloroplast of the green alga Chlamydomonas reinhardtii, the mechanism under study may well occur in many organisms.Broader impacts. This project has potential for broad biological impacts, and will also be used as a training opportunity. The major novelty is that the form of protein splicing under study is newly described, and therefore suggests a previously unrecognized mechanism for creating diversity in proteins. This research may therefore stimulate or facilitate exploration of protein splicing on a larger scale, i.e. in other organisms. If this type of protein splicing is widespread, there will be important implications for analyzing the current large datasets of RNA and protein sequences, which in turn impinges on the analysis of the ever-growing deluge of genomic information. The project will train students at the high school and undergraduate levels, including those from underrepresented groups, through an internship program run at the host institution (Boyce Thompson Institute). This program has a strong record of diverse participation, and ensures through strong management a broad exposure to plant science, as well as career-related guidance and perspective. The project will create a video publication on a key biochemical technique employed in this project; a prior video was widely viewed and has assisted re-searchers in many laboratories. Boyce Thompson Institute has committed resources to a robust training environment not only for the next generation of scientists, but also for high school teachers. In addition, it will commit resources to support programmatic development for a variety of outreach programs. These programs provide numerous opportunities for postdoctoral fellows at Boyce Thompson Institute, including those to be trained in this project, to participate directly in outreach efforts and thus enrich their career paths.

可以从指定它的RNA的蛋白质序列预测。换句话说，嵌入RNA分子中的代码与该RNA活性作为模板产生的蛋白质之间通常存在对应关系。一种称为RB47的蛋白质无法遵循此范式，是该项目的主要重点。具体而言，RB47是根据其RNA模板合成的，但随后去除了蛋白质的内部部分，将两个侧翼部分合并为一个剪接的较小蛋白质。这种蛋白质剪接反应在本质上是已知的，但通常是自发的。在RB47的情况下，需要特定的细胞因子来剪接它。这项研究的重点是该剪接的机制，这没有明显的先例。 RB47的第二个方面是该蛋白质的初始版本是酶惰性的，但是剪接的版本具有RNA裂解活性。因此，蛋白质剪接激活该酶。这暗示剪接也具有调节功能。该项目具有三个特定的目标：首先，定义RNA裂解活性所需的蛋白质本身中的信号；其次，要发现进行剪接反应的分子仪。第三，使用遗传实验来辨别RB47的生物学作用。尽管RB47是绿色藻类衣原体Reinhardtii的叶绿体中的一种蛋白质，但研究的机制可能会在许多生物体中发生。该项目具有广泛的生物学影响，也将被用作培训机会。主要的新颖性是，新描述的蛋白质剪接形式是新描述的，因此提出了一种以前未经认可的机制来创造蛋白质多样性。因此，这项研究可能会刺激或促进更大范围的蛋白质剪接的探索，即在其他生物体中。如果这种类型的蛋白质剪接是广泛的，那么分析当前的RNA和蛋白质序列的大型数据集将具有重要意义，这反过来又影响了不断增长的基因组信息洪水泛滥。该项目将通过在主持机构（Boyce Thompson Institute）进行的实习计划来培训高中和本科级别的学生，包括来自代表性不足的团体的学生。该计划具有多种参与的记录，并通过强大的管理对植物科学以及与职业相关的指导和观点确保。该项目将创建有关该项目采用的关键生化技术的视频出版物；先前的视频被广泛观看，并协助了许多实验室的重新搜索者。博伊斯·汤普森学院（Boyce Thompson Institute）不仅为下一代科学家，而且对高中教师提供了强大的培训环境，为培训环境提供了资源。此外，它将投入资源，以支持各种外展计划的程序化开发。这些计划为博伊斯·汤普森学院（Boyce Thompson Institute）提供了许多机会，包括接受该项目的培训的人，直接参与外展工作，从而丰富他们的职业道路。