Six miR-34/449 miRNAs derived from three genomic loci constitute the most abundant miRNAs expressed in airway multiciliated cells (MCCs), indicating a functional redundancy of this miRNA family in these cells. Removal of the redundancy by deletion of all three miRNA genes in mice leads to defective motile ciliogenesis. In mir-34/449 triple knockout (TKO) mice, faulty cilia compromise airway mucociliary clearance, giving rise to the congestion of mucus and infection in the upper respiratory tract. Notably, severe respiratory distress causes early postnatal mortality in around half of TKO mice. Respiratory distress and infertility phenotypes displayed in survived adult TKO mice resemble the key symptoms of human Primary Ciliary Dyskinesia (PCD). Knockdown of Cp110, a major miR-34/449 target, can largely rescue motile ciliogenesis in mir-34/449 deficient MCCs. Overall, miR-34/449 miRNAs represent the very first non-coding RNAs whose deficiency in mice causes PCD. This proposal builds on these previous findings to further study miR-34/449 miRNAs in airway MCCs. During the K99 phase of the award, I will learn three cutting-edge technologies in life science: super- resolution structured illumination microscopy (SIM) imaging, next generation RNA-sequencing (RNA-seq) and related bioinformatics analysis, and CRISPR/Cas9 genome editing. Coursework and collaboration with leading scientists in these fields will help me master these techniques, which will facilitate not only completion of this proposed project but also my long term scientific career.
Aim 1 will explore the cellular defects of TKO MCCs. 3D SIM live imaging will be used to visualize the dynamics of GFP/RFP tagged basal bodies and intraflagellar transport during motile ciliogenesis in cultured control and TKO MCCs. These studies will reveal hidden cellular defects in real time during ciliogenesis in TKO MCCs, which are missed by the endpoint examination of motile cilia in our previous work. Additionally, using straight knockout mice in our previous study may also blind us on the potential functions of miR-34/449 miRNAs in cilia maintenance after MCC maturation. To probe this, conditional deletion of mir-34/449 genes in mice after MCC maturation will be used in Aim 1. The first part of Aim 2 will identify the multiple targets regulated by miR-34/449 miRNAs in MCCs. RNA-seq-initiated unbiased approaches as well as biased candidate approaches will be used to uncover additional miR-34/449 targets in MCCs. These studies are intended to be complete before the R00 phase. The second part of Aim 2 will evaluate miR-34/449 targets in vivo in TKO mice and in primary human airway epithelial cells (HAECs). Reinforced miR-34/449 expression and stable knockdown of miR-34/449 targets like Cp110 in airway epithelia of TKO mice will determine whether these procedures can rescue motile cilia in vivo and further relieve respiratory distress in TKO mice. The functional readouts of miR-34/449-mediated target repression in primary HAECs will be studied by CRISPR/Cas9 editing to disrupt the miR-34/449 binding sites in the 3'UTR of target mRNA like CP110. These experiments serve as proof-of-principal studies to test the feasibility of using miR- 34/449 miRNAs and their targets to treat ciliary disease, and will evaluate the extent to which mouse studies can be applied to human biology.
Aim 3 will investigate the biology of the unique apical localization of miR- 34/449 miRNAs in MCCs. Sequence mutation assays and a novel Csy4-based RNA-pull method will be used to identify the sequence zip codes inside miRNAs and the protein carriers that direct miR-34/449 apical transport in MCCs. The localization study will be extended to human MCCs as well to fully understand the conservation of miR-34/449 miRNAs in MCCs in terms of sub-cellular location and its underlying mechanisms. Overall, this proposal will generate a comprehensive and detailed picture of miR-34/449 miRNAs in mouse and human airway MCCs, which will have significant implications on both basic biology and potential clinical applications.
Deficient expression of miR-34/449 miRNAs is associated with a variety of respiratory diseases, including PCD, asthma, and chronic obstructive pulmonary disease (COPD). A deeper understanding of miR-34/449 miRNAs in airway MCCs will support the development of diagnoses and treatment strategies for these motile cilia-related lung diseases.
