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Bridges et al. discuss current knowledge, lingering questions, and emerging themes surrounding lncRNA subcellular localization and its impact on cellular functions., Subcellular localization of RNAs has gained attention in recent years as a prevalent phenomenon that influences numerous cellular processes. This is also evident for the large and relatively novel class of long noncoding RNAs (lncRNAs). Because lncRNAs are defined as RNA transcripts >200 nucleotides that do not encode protein, they are themselves the functional units, making their subcellular localization critical to their function. The discovery of tens of thousands of lncRNAs and the cumulative evidence involving them in almost every cellular activity render assessment of their subcellular localization essential to fully understanding their biology. In this review, we summarize current knowledge of lncRNA subcellular localization, factors controlling their localization, emerging themes, including the role of lncRNA isoforms and the involvement of lncRNAs in phase separation bodies, and the implications of lncRNA localization on their function and on cellular behavior. We also discuss gaps in the current knowledge as well as opportunities that these provide for novel avenues of investigation.

With the realization that mechanical forces mediate many biological processes and contribute to disease progression, researchers are focusing on developing new methods to understand the role of mechanotransduction in biological systems. Despite recent advances in stretching devices that analyze the effects of mechanical strain in vitro, there are still possibilities to develop new equipment. For example, many of these devices tend be expensive, whereas few have been designed to assess the effects of mechanical strain driven by the extracellular matrix (ECM) to epithelial cell monolayers and to cell-cell adhesion. In this chapter, we introduce a cost-efficient, user-friendly, 3D-printed stretching device that can be used to test the effects of mechanical strain on cultured epithelial cells. Evaluation of the device using speckle-tracking shows homogeneous strain distribution along the horizontal plane of membranes at 2.5% and 5% strains, supporting the reliability of the device. Since cell-cell junctions are mechanosensitive protein complexes, we hereby used this device to examine effects on cell-cell adhesion. For this, we used colon epithelial Caco2 cell monolayers that well-differentiate in culture and form mature adherens junctions. Subjecting Caco2 cells to 2.5% and 5% strain using our device resulted in significant reduction in the localization of the core adherens junction component E-cadherin at areas of cell-cell contact and its increased translocation to the cytoplasm, which in agreement with other methodologies showing that increased ECM-driven strain negatively affects cell-cell adhesion. In summary, we here present a new, cost-effective, homemade device that can be reliably used to examine effects of mechanical strain on epithelial cell monolayers and cell-cell adhesion, in vitro.

E-cadherin is the core component of epithelial adherens junctions, essential for tissue development, differentiation, and maintenance. It is also fundamental for tissue barrier formation, a critical function of epithelial tissues. The colon or large intestine is lined by an epithelial monolayer that encompasses an E-cadherin-dependent barrier, critical for the homeostasis of the organ. Compromised barriers of the colonic epithelium lead to inflammation, fibrosis, and are commonly observed in colorectal cancer. In addition to its architectural role, E-cadherin is also considered a tumor suppressor in the colon, primarily a result of its opposing function to Wnt signaling, the predominant driver of colon tumorigenesis. Beyond these well-established traditional roles, several studies have portrayed an evolving role of E-cadherin as a signaling epicenter that regulates cell behavior in response to intra- and extra-cellular cues. Intriguingly, these recent findings also reveal tumor-promoting functions of E-cadherin in colon tumorigenesis and new interacting partners, opening future avenues of investigation. In this Review, we focus on these emerging aspects of E-cadherin signaling, and we discuss their implications in colon biology and disease.

The RNA interference (RNAi) machinery is an essential component of the cell, regulating miRNA biogenesis and function. RNAi complexes were thought to localize either in the nucleus, such as the microprocessor, or in the cytoplasm, such as the RNA-induced silencing complex (RISC). We recently revealed that the core microprocessor components DROSHA and DGCR8, as well as the main components of RISC, including Ago2, also associate with the apical adherens junctions of well-differentiated cultured epithelial cells. Here, we demonstrate that the localization of the core RNAi components is specific and predominant at apical areas of cell-cell contact of human normal colon epithelial tissues and normal primary colon epithelial cells. Importantly, the apical junctional localization of RNAi proteins is disrupted or lost in human colon tumors and in poorly differentiated colon cancer cell lines, correlating with the dysregulation of the adherens junction component PLEKHA7. We show that the restoration of PLEKHA7 expression at adherens junctions of aggressively tumorigenic colon cancer cells restores the junctional localization of RNAi components and suppresses cancer cell growth in vitro and in vivo. In summary, this work identifies the apical junctional localization of the RNAi machinery as a key feature of the differentiated colonic epithelium, with a putative tumor suppressing function.

Colon cancer is the third most common and the second deadliest type of cancer in the United States. A typical characteristic of colon tumors is the compromised integrity of the colonic epithelial monolayer. The Adherens Junction (AJ) is a cell-cell adhesion complex essential for the maintenance of the epithelial integrity, composed of E-cadherin and the catenin family of proteins. We have recently shown that the E-cadherin-p120 catenin partner PLEKHA7 is critical for epithelial monolayer integrity. We have also revealed that PLEKHA7 recruits the core components of the RNA interference (RNAi) machinery, such as Ago2, DROSHA and DGCR8, as well as miRNAs at the mature apical adherens junctions, to suppress expression of pro-tumorigenic markers, through miRNA-mediated silencing. However, we still haven’t examined the extend of the contribution of this mechanism to colon tumorigenesis and its modes of regulation. We hypothesize that the adherens junction-RNAi mechanism acts as an epithelial sensor that tethers monolayer integrity to cell behavior. To examine this, we generated 3D cultures of well-differentiated Caco2 colon epithelial cells on Matrigel to examine the distribution of RNAi proteins. Indeed, co-localization of PLEKHA7 with RNAi proteins occurs specifically at the apical surface of these spheroids, whereas PLEKHA7 depletion results in loss of junctional localization of RNAi proteins and in enlarged, multilayered structures. Corroborating these results, PLEKHA7 and RNAi components co-localize specifically at the apical surface of normal colon crypts, whereas extensive disruption or loss of this co-localization was observed in colon tumor patient samples. Fibrosis and extensive deposition of extracellular matrix (ECM) due to chronic inflammation is a precursor to colon cancer. ECM can affect adherens junctions integrity, through integrin-Src signaling. Indeed, we have found that Src activity disrupts localization of PLEKHA7 and RNAi proteins to the junctions. To investigate the potential role of ECM in the regulation of the junctional RNAi mechanism, we are now examining Caco2 cells grown on different ECM substrates, as well as under different conditions of mechanical strain, since fibrosis poses mechanical stress on cells. In summary, our data point towards a novel putative tumor suppressor mechanism, of which we are investigating its modes of regulation and its involvement in colon tumorigenesis. Citation Format: Amanda C. Daulagala, Catherine Bridges, Lauren Rutledge, Joyce Nair-Menon, Micheal Yost, Antonis Kourtidis. Regulation and functional role of the cell-cell junction-associated RNAi machinery in colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 64.