Activation of an Endoribonuclease by Non-intein Protein Splicing

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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.