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.

蛋白质的序列可以从指定它的RNA的序列中预测出来，换句话说，嵌入RNA分子中的密码子和以该RNA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RB 47的蛋白质未能遵循这种范式，是该项目的主要焦点。具体地说，RB 47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一个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。RB 47的剪接需要特定的细胞因子，目前的研究主要集中在其剪接机制上，尚无明显的先例。RB 47的第二个方面是蛋白质的初始形式是酶惰性的，但剪接形式具有RNA切割活性。因此，蛋白质剪接激活这种酶。这暗示剪接也具有调节功能。该项目有三个具体目标：第一，确定RNA切割活性所需的蛋白质本身的信号;第二，发现进行剪接反应的分子装置;第三，使用遗传实验来识别RB 47的生物学作用。虽然RB 47是一种来自绿色莱茵衣藻叶绿体的蛋白质，但所研究的机制可能会发生在许多生物体中。该项目有可能产生广泛的生物影响，也将被用作培训机会。主要的新奇在于，研究中的蛋白质剪接形式是新描述的，因此表明了一种以前未被认识到的蛋白质多样性产生机制。因此，这项研究可能会刺激或促进更大规模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普遍的，将有重要的影响，分析目前的大型数据集的RNA和蛋白质序列，这反过来又影响到分析不断增长的洪水的基因组信息。该项目将通过在东道机构（博伊斯汤普森研究所）实施的实习方案，培训高中和大学生，包括来自代表性不足群体的学生。该计划具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。该项目将创建一个视频出版物的关键生化技术在这个项目中使用;以前的视频被广泛观看，并已协助许多实验室的研究人员。博伊斯汤普森研究所已承诺资源，以一个强大的培训环境，不仅为下一代的科学家，而且为高中教师。此外，它将投入资源，支持各种外联方案的方案发展。这些项目为博伊斯汤普森研究所的博士后研究员提供了许多机会，包括那些在这个项目中接受培训的人，直接参与外联工作，从而丰富他们的职业道路。