PROJECT SUMMARY Compromised epithelial integrity is a hallmark of gastrointestinal abnormalities, such as inflammatory bowel disease and colon cancer, which is the third most prevalent and second most lethal form of cancer. The high incidence rates of these diseases suggest that we still don’t fully understand the underlying mechanisms. Recently, we discovered a mechanism that links epithelial tissue integrity with the RNA interference (RNAi) machinery and with miRNA regulation. We have shown that the adherens junctions, which is an essential architectural component of the cell, recruit the microprocessor and the RNAi-induced silencing complex (RISC), the core components of the RNAi machinery, as well as a specific set of miRNAs, in colon epithelial cells. This interaction occurs through PLEKHA7, a novel partner of the E-cadherin cell-cell adhesion complex. PLEKHA7 loss results in compromised epithelial integrity, decreased levels and function of a set of miRNAs and in increased anchorage-independent growth, an indicator of epithelial transformation. Our preliminary data show extensive mis-localization or loss of PLEKHA7 in colon cancer cell lines and tumor patient samples. Interestingly, an RNA-CLIP experiment followed by RNA sequencing revealed association of PLEKHA7 with long non-coding RNAs (lncRNAs). LncRNAs can interact with miRNAs and the RNAi machinery in multiple ways and a number of them has been implicated in intestinal diseases. However, our knowledge on lncRNA regulation and function is still limited. Our preliminary data show that PLEKHA7 loss results in altered expression of a number of these lncRNAs, including upregulation of MIR17HG, a known promoter of cellular transformation. We hypothesize that the adherens junctions recruit and regulate the RNAi machinery and lncRNAs to maintain colon epithelial homeostasis. We will test our hypothesis in two Aims that will determine whether: 1) PLEKHA7 suppresses MIR17HG levels through RISC and miRNAs at adherens junctions; 2) PLEKHA7 maintains the normal colon epithelial phenotype through miRNAs and MIR17HG. This study is significant, since it provides a missing mechanistic link between epithelial architecture and cell behavior and a new unexpected localized regulation of RNAi and lncRNAs. The proposed work is innovative, because it tethers two previously unrelated fields, cell-cell adhesion and non-coding RNA biology. The impact of the study is that it will advance our understanding of the underlying mechanistic causes of intestinal diseases and will lay the foundation for the systematic interrogation of the newly discovered connection between the adherens junctions, the RNAi machinery and non-coding RNAs.