Intellectual Merit: Plant organelles convert certain cytidines to uridines in mRNA post-transcriptionally in a process known as RNA editing. This process is critical for proper expression of many chloroplast and mitochondrial genes that have accumulated T to C mutations. If not edited, these genes would produce non-functional proteins that would have a detrimental effect on important processes such as photosynthesis and respiration. The complete composition of the plant RNA editing protein complexes, the plant editosomes, is unknown. The selection of the C to be converted to U is performed by Pentatricopeptide Repeat-Motif (PPR) RNA-binding proteins, which recognize sequences on the RNA in close proximity to the C target. In addition to the large PPR protein family, members of two additional plant protein families, the RIP and ORRM families, have recently been found to be required for RNA editing in organelles. Unlike the site-specific PPR proteins, which are needed for editing of only one or a few individuals Cs, the ORRM and RIP proteins are essential for editing efficiency of the majority of edited Cs in chloroplasts and/or mitochondria and thus likely are components of most editosomes. The role of additional ORRM family members will be examined by mutant analysis and gene silencing in conjunction with a deep sequencing editing assay. ORRM and RIP proteins will be expressed with tandem affinity purification tags in transgenic plants in order to isolate editosomes, whose component proteins will then be identified by mass spectrometric methods.
Broader Impacts: C-to-U RNA editing is able not only to change the encoded amino acid, but can also create start and stop codons, profoundly affecting the accumulation and function of proteins. Understanding the site-specific plant RNA editing mechanism will pave the way for future genetic engineering of plants and other organisms through deliberate post-transcriptional modification of codons. The project will provide training opportunities for both graduate and undergraduate students, including ones from other colleges who are participating in a summer internship program that offers research experience and formal lectures. The PI will continue her efforts to promote the recruitment, retention, and advancement of women in STEM fields. The coPI will disseminate a detailed deep sequencing protocol that is valuable not only for RNA editing researchers, but more generally for scientists studying a subset of the RNA transcriptomes in their experimental organisms.
