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.

蛋白质的序列可以根据指定它的RNA的序列来预测。换句话说，RNA 分子中嵌入的代码与以该 RNA 作为模板的活性产生的蛋白质之间通常存在对应关系。一种名为 RB47 的蛋白质未能遵循这一范式，但却是该项目的主要焦点。具体来说，RB47是根据其RNA模板合成的，但随后蛋白质的内部部分被去除，两个侧翼部分被组合成剪接的、更小的蛋白质。这种蛋白质剪接反应在自然界中是已知的，但通常是自发的。就 RB47 而言，需要特定的细胞因子来对其进行剪接。研究主要集中在这种剪接的机制上，目前还没有明显的先例。 RB47 的第二个方面是该蛋白的初始版本是酶惰性的，但剪接版本具有 RNA 切割活性。因此，蛋白质剪接会激活这种酶。这暗示剪接也具有调节功能。该项目有三个具体目标：首先，定义蛋白质本身内 RNA 裂解活性所需的信号；其次，发现进行剪接反应的分子装置；第三，利用基因实验来辨别RB47的生物学作用。尽管RB47是来自绿藻莱茵衣藻叶绿体的蛋白质，但正在研究的机制很可能发生在许多生物体中，并产生更广泛的影响。该项目具有广泛的生物影响潜力，也将用作培训机会。主要的新颖之处在于，新描述了正在研究的蛋白质剪接形式，因此提出了一种以前未被认识的创造蛋白质多样性的机制。因此，这项研究可能会刺激或促进更大规模（即在其他生物体中）蛋白质剪接的探索。如果这种类型的蛋白质剪接广泛存在，将对分析当前的大型 RNA 和蛋白质序列数据集产生重要影响，这反过来又会影响对不断增长的大量基因组信息的分析。该项目将通过在主办机构（博伊斯汤普森研究所）开展的实习计划，培训高中和本科生，包括来自代表性不足群体的学生。该计划拥有多元化参与的良好记录，并通过强有力的管理确保广泛接触植物科学以及与职业相关的指导和观点。该项目将制作一个关于该项目中采用的关键生化技术的视频出版物；之前的一段视频被广泛观看，并为许多实验室的研究人员提供了帮助。博伊斯汤普森研究所投入资源，不仅为下一代科学家，也为高中教师提供强大的培训环境。此外，它将投入资源来支持各种外展计划的计划开发。这些项目为博伊斯汤普森研究所的博士后研究员提供了大量机会，包括那些在该项目中接受培训的人，直接参与外展工作，从而丰富他们的职业道路。