Date of Award

2022

Embargo Period

4-26-2024

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Regenerative Medicine and Cell Biology

College

College of Graduate Studies

First Advisor

Antonis Kourtidis

Second Advisor

Stephen Duncan

Third Advisor

Robin Muise-Helmericks

Fourth Advisor

Viswanathan Palanisamy

Fifth Advisor

Je-Hyun Yoon

Abstract

Adherens junctions (AJs) are architectural components of the cell that are essential for epithelial tissue integrity. These adhesive structures tether intracellularly to an apical actin ring to form the zonula adherens (ZA). Our previous work revealed that PLEKHA7, a E-cadherin-p120 catenin partner at the ZA, recruits the microprocessor, the RNA-induced silencing complex (RISC), and distinct sets of miRNAs and mRNAs specifically to the ZA. There, the processing of and silencing by a set of miRNAs suppresses oncogenic mRNA expression. PLEKHA7 depletion results in compromised epithelial integrity, altered miRNA function, and pro-tumorigenic cell transformation; we have found extensive dysregulation of this mechanism in colon cancer. Notably, we have also identified lncRNAs associated with PLEKHA7, though the functional significance of junctional lncRNA localization has not been explored. Here, we examined the regulation of lncRNAs by this PLEKHA7-associated RNAi complex and its implications for cellular homeostasis. We also addressed a critical remaining question: why are these RNAi complexes specifically recruited to the ZA? RNA-sequencing revealed that PLEKHA7 regulates the levels of a set of lncRNAs. The most significantly upregulated junction-associated lncRNA following PLEKHA7 depletion was a previously unrecognized short isoform of the MIR17HG lncRNA, which we named MIR17HG_S. Through mechanistic interrogation, we demonstrate that PLEKHA7 suppresses MIR17HG_S levels via the junctional RISC, specifically AGO2 and miR-203a. MIR17HG_S overexpression promoted cellular transformation, whereas expression analysis revealed that MIR17HG_S is significantly upregulated in patient colon tumor tissues. These findings identify a novel localized mechanism of lncRNA regulation at the ZA and identify MIR17HG_S as a pro-tumorigenic lncRNA, downstream of disrupted junctional RNAi. Further, we demonstrate ZA-specific recruitment of AGO2 depends on both the structural and tensile integrity of the actomyosin cytoskeleton. We show that PLEKHA7 and AGO2 interact with three LIM domain-containing proteins, namely LMO7, LIMCH1, and PDLIM1, all of which regulate actomyosin organization and consequently mediate AGO2 ZA recruitment. Together, this work identifies a mechanosensitive RNAi complex at the ZA that regulates lncRNAs with critical roles in cell behavior, such as MIR17HG_S. Future work will address how distinct mechanosensitive cues may be regulating non-coding RNA function and influencing cell behavior, not only in diseases, like cancer, but also in physiological processes that require actin remodeling, such as tissue morphogenesis.

Rights

All rights reserved. Copyright is held by the author.

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