Project Summary Sequence alterations that change the genome-encoded information present in RNAs, referred to as RNA editing, provide a powerful way to diversify the transcripts expressed in an organism’s tissues over time. The objective of the parent research proposal is to determine molecular mechanisms of how RNA binding proteins influence substrate recognition by the ADAR RNA editing enzymes. ADARs catalyze millions of adenosine (A) to inosine (I) modifications in eukaryotic transcriptomes to play an essential role in the creation of proteomic and phenotypic diversity. Despite the prevalence of A-to-I editing, there is a gap in knowledge of how ADARs edit specific adenosines to varying degrees during development and in specific cell types. My lab has made significant contributions to this outstanding question by identifying roles for naturally occurring editing-deficient ADAR proteins in regulating editing. The proposed research takes an integrated approach using both the model organism, Caenorhabditis elegans, and human glioblastoma (brain tumor) cell lines with a combination of biochemistry, genomics, and molecular biology to connect the molecular mechanisms of RNA recognition by ADARs to functional consequences on RNA editing and gene expression. Our main goals are to define the molecular mechanism of how an editing-deficient ADAR protein can recruit the RNA editing enzyme to specific adenosines in vivo, dissect the mechanism of how certain neural transcripts are selectively edited and to determine the cellular targets and impact of the editing-deficient human ortholog on the glioblastoma transcriptome. An essential approach used in our work is the generation and biochemical/genomic analysis of transgenic C. elegans. This is a powerful system as loss of ADARs results in behavior/neuronal phenotypes in C. elegans in contrast to lethality in mice. Therefore, we can mechanistically dissect the process of ADAR editing at specific sites by using genetic mutants in both the RNA editing enzyme and the editing-deficient ADAR protein. In addition, using fluorescent proteins fused to ADAR substrates, we can determine molecular features critical for editing of specific adenosines in vivo. In this supplemental proposal, I am requesting funds for the replacement of my aging fluorescence microscope that is used to both prepare our transgenic worms for experiments and screen the creation of additional transgenic strains for the proposed research. While this instrument has served us well and is still used, the repairs needed, and age of the system have indicated that it is near the end of its lifetime. The purchase of a Zeiss Discovery V20 fluorescence dissecting microscope will enable us to perform the proposed research, and the dedicated use of this instrument by my laboratory will increase experimental output to meet the goals of determining the molecular mechanisms that regulate RNA editing in vivo.