! 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. Loss of these modifications results in lethality in mice and behavioral phenotypes in worm and fly model systems. Consistent with an important role in both normal development and proper neuronal function, aberrant RNA editing has been linked over 35 human pathologies, including several neurological disorders, metabolic diseases, and cancer. Despite the significance of A-to-I editing, there is a gap of knowledge in the molecular mechanisms that regulate editing. Our long-term goal is to understand how differing modes of RNA recognition by ADARs, the enzymes responsible for A-to-I RNA editing, result in specific effects on RNA editing to allow for the rational development of therapeutics that can alter ADAR function, thus improving human health. Towards this goal, we have identified cellular roles for naturally occurring editing-deficient ADAR family members in altering RNA editing in worms and humans. The objective of this proposal is to determine the molecular mechanisms of how these regulators influence recognition of specific transcripts by the editing enzymes. The central hypothesis of the proposed research is that RNA recognition by editing-deficient ADAR family members can both promote and antagonize RNA recognition by ADAR enzymes, and both of these functions are critical for maintaining proper editing levels in vivo. This hypothesis has been formulated, in large part, on our findings that ADR-2, the only A-to-I editing enzyme in C. elegans, functions with ADR-1, an editing-deficient ADAR family member, to edit across the transcriptome, including a transcript required for proper neuronal function. However, ADR-1 is not required for ADR-2 to edit all adenosines. In fact, ADR-1 inhibits editing of certain neural mRNAs, supporting our hypothesis that editing-deficient ADARs serve as both positive and negative regulators of editing depending upon the transcript. This regulatory function is likely evolutionarily conserved, as we recently demonstrated that editing-deficient human ADAR3 binds a critical neuronal transcript to alter editing in glioblastoma.
In Aim 1, a RNA immunoprecipitation approach, which has been established as feasible in the applicants? hands, will be combined with high-throughput sequencing and genetic mutants the applicant developed and characterized to determine how ADR-1 recruits the editing enzyme to specific targets.
In Aim 2, a cutting-edge neural isolation technique will be coupled to high-throughput sequencing to dissect the mechanism of how certain neural transcripts are selectively edited.
In Aim 3, the cellular targets of an inactive editor, human ADAR3, will be identified and the impact of ADAR3 on the proper balance of unedited and edited transcripts will be determined. The proposed research is significant, because the identified factors and regulatory mechanisms are likely to provide new targets for therapeutic interventions, in addition to fundamentally advancing the fields of RNA editing and dsRNA mediated cellular pathways.
! Alterations in RNA editing occur in over 35 human pathologies, including several neurological disorders, metabolic diseases, and cancer, but these changes are often uncorrelated with levels of the editing enzymes, underscoring the need to identify cellular factors that regulate editing. The proposed research takes an integrated approach using C. elegans and human neural cell lines to determine both the molecular features required for editing of specific adenosines in vivo and detailed molecular mechanisms of how cellular factors regulate RNA recognition by editing enzymes. This fundamental knowledge will represent a critical first step in developing therapeutics that modulate editing and improve human health.