A novel mouse model to distinguish the specific physiological significance of RNAi and biophysical mechanisms of microRNA - Project Summary MicroRNAs (miRs) are evolutionally conserved small non-coding RNA molecules and control most biological events, including apoptosis, cell proliferation, metabolism, cell fate determination, organogenesis, development, stress responses, and tumorigenesis. Classically, miRs are known to negatively regulate gene expression through RNA interference (RNAi) mechanism. Recently, we revealed a novel biophysical action of miR1, which is the most predominant miR in the heart and is downregulated in human heart failure. We discovered that miR1directly binds to inward rectifier potassium channel Kir2.1, resulting in direct suppression of the IK1 current and leading to biophysical modulation of cardiomyocyte cellular electrophysiological functions. Our studies suggest that miR1 modulates the development and homeostasis of tissues/organs through two different mechanisms: the immediate effect (seconds to minutes) of newly-discovered biophysical modulation and long-term effect (hours to days) of RNAi. With this important new finding, it now becomes essential to understand how these two distinct miRs mechanisms of action coordinate to regulate the development and homeostasis of our body. However, there is no valid model that can distinguish the specific physiology significance of biophysical modulation versus RNAi mechanism. We found that an arrhythmia-associated hSNP14A/G specifically defects the biophysical action while maintaining miR1’s RNAi function; therefore, we propose to develop a unique transgenic mouse model that can separate the specific contribution coming from the biophysical modulation and dissect the pure contribution of RNAi in maintain the homeostasis of multi organs/systems. We will develop miR1-full-KO/muscle-specific inducible hSNP14A/G-knock-in mice, and we hypothesize that an expression of hSNP14A/G in muscle cells could rescue the postnatal lethality of miR1-full- KO mice. We will investigate if lacking biophysical function of hSNP14A/G induces any abnormal phenotypes (Aim 1), such as arrhythmia, heart failure, and abnormal contractility of skeletal muscle, which will demonstrate the specific role of miR1’s biophysical modulation in regulation of the homeostasis in vivo. We will also turn off the expression of hSNP14A/G by administration of doxycycline and investigate the specific physiological significance of miR1’s RNAi mechanism in the heart (Aim 2). This unique animal model will be very valuable to investigate the critical role of miR1 in multiple organs/systems, including the heart, skeletal muscle, various types of cancers. Understanding the specific contributions of miR’s biophysical modulation and RNAi in vivo will expand the biological significance of miRs and guide us to develop new therapeutic approaches for human diseases through targeting of miRs.
