Molecular mechanisms that regulate ADAR target recognition and RNA editing in vivo

Trustees Of Indiana University, Bloomington IN

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Abstract

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. Over the last year, we have made exceptional progress on understanding the in vivo RNA binding sites of C. elegans ADR-1 (the deaminase-deficient ADAR) and ADR-2 (the editing enzyme). In this supplemental proposal, I am requesting funds to support the salary and training of a post-baccalaureate student that is from an ethnic group that is underrepresented in health-related sciences within the United States. This individual will perform experiments related to goals of the parent grant while also receiving direct mentoring in academic research and core professional skills, which will enable her to be competitive to enter graduate training wherein she will help to increase the Hispanic physician scientist population, which currently only make up 5.8% M.D./Ph.D. graduates in the US. The post-baccalaureate researcher will specifically work on three different aims building from our recent preliminary data. She will 1) confirm novel RNA binding targets of ADR-1 and ADR-2 using RNA immunoprecipitation coupled to quantitative real-time PCR (RIP-qPCR), 2) examine the impact of ADR-1 binding on gene expression using qPCR and 3) design in vivo reporter models to examine how loss of ADR-1 binding sites influences editing. Completion of any one of the goals over the next year will further our understanding of the role of non-editing ADAR family proteins in gene regulation, results of which will be shared in research conferences and publications.