Functional Decoding of Development-Associated Rna Editomes---Skeletal Myogenesis as a Research Model

RNA-centric single nucleotide polymorphisms are prevalent in the mammalian transcriptomes. These so-called RNA editing events, majority of which are of the A-to-I conversion 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. However, among mechanisms that demarcate the transcriptome, RNA editing remains one of the less resolved steps – what target pre-mRNAs undergo editing-induced sequence recoding, what mode of regulation is achieved molecularly, how the RNA editing events are decoded at the proteome and cellular levels, and how the RNA editome is coordinated with its developmental role remain to be answered. To this end, our previous deep sequencing-based, transcriptome-wide studies have catalogued accurate and comprehensive profiles of the human and monkey RNA editomes, and uncovered the evolutionary significance of this altered encoded information and a potential functional crosstalk with other post-transcriptional processes. By extending these systems findings to mechanistic understanding, our recent efforts have also unveiled novel tissue-specific but divergent roles of the RNA editors – ADAR1 and ADAR2 – in skeletal myogenesis, a well-orchestrated multistep differentiation program through which precursor myoblast cells mature into multinucleated myotubes. Focusing on this particular spatiotemporal context, the current study is aimed to further delineate the regulation and developmental significance of ADAR2-associated RNA editome. To provide a systematic investigation into this novel aspect of differentiation-associated gene regulation, we will employ an integrated approach that entails functional genomics, cell-based, and animal studies. First, we will perform a series of sequence-based experiments to decode the RNA editome and its proteomic outcome (Aim 1). Next, the underlying molecular mechanism and cellular consequence of ADAR2-associated RNA editome will be thoroughly dissected using biochemical and cell biological experiments in a myoblast system (Aim 2). Finally, “gain-of-editing” and “loss-of-editing” knock-in mouse models will be established, via the CRISPR/Cas system, for phenotypic characterization of functional outcome of the recoding events in the context of muscle development and physiology (Aim 3). A powerful aspect of our proposed studies is the integrated experimental paradigm that is easily adaptable to additional RNA editing-related studies as well as other mutation-centric conditions, such as cancer and hereditary diseases. By establishing the regulatory network of ADAR2 and its physiological role, another significant implication of these studies is the tremendous potential for the development of new therapeutic schemes for muscle-specific diseases or other conditions caused by defective ADAR and/or RNA editing.

Project ID:PC10501-2222
External Project ID:MOST104-2320-B182-029-MY3