Functional Role of RNA Editing Enzyme ADAR in Transcriptome Landscaping and Its Developmental Relevance

Widespread occurrence of RNA-centric single nucleotide polymorphisms has been reported in the mammalian transcriptomes. These so-called RNA editing events, majority of which are of the A-to-I type and catalyzed by the double-stranded RNA-specific adenosine deaminse acting on RNA (ADAR) family proteins, constitute an integral step in generating the diversity and plasticity of cellular RNA signatures. Among mechanisms that demarcate the transcriptome, RNA editing remains one of the less resolved steps in terms of regulation and functional consequence. Despite extensive documentation of edited sites through early bioinformatic approaches and the enzymatic and functional characterization of the ADAR proteins, the complete catalog of direct substrates, as well as the corresponding functional consequences, is not yet fully compiled. Also lacking in this regard are data for an explicit implication of this factor in other aspects of gene expression regulation and detailed characterization of its developmental relevance, particularly in spatiotemporal contexts. Owing to the sequence-specific nature and extent of the outcome of RNA editing, global and unequivocal identification of RNA editing targets requires complete information on transcriptome sequences, and thus a necessarily high throughput and base resolution of the experimental approach undertaken. The use of the next-generation, whole transcriptome deep-sequencing platform thus constitutes an important basis of this research, with which we aim to address the mode of action of the ADAR proteins in shaping the cellular transcriptome. In this proposal, we are attempting to outline and execute a set of synergistic experiments to further explore the role of ADAR enzymes and to provide transcriptome-wide insights into the mechanisms and molecular consequences of RNA editing. First, we will establish a comprehensive profile of the editing and transcript targets of ADAR via RNA-Seq and small RNA-Seq. Bioinformatic tools will be applied to reveal the complement of RNA transcripts in terms of structure and quantity, facilitating cross-sample comparison for the unbiased identification of targets. Next, the underlying molecular mechanism and cellular consequence of ADAR’s functions will be thoroughly dissected via biochemical and cell biological experiments. Finally, conditional knockout mice model will be established for phenotypic characterization of functional outcome of ADAR-mediated activity in the context of tissue differentiation and development. A powerful aspect of our proposed studies is the interplay between the functional genomic approach and the cell-based and knockout mice tools that will allow us to directly and comprehensively address the roles of ADAR in transcriptome regulation and developmental processes, and any interconnection thereof. Another significant implication of our research lies in the establishment of an experimental paradigm that can be extended to functional characterization of ADAR proteins in other physiological contexts or post-transcriptional regulators in general.

Project ID:PC10301-0903
External Project ID:NSC101-2320-B182-036-MY3