Dynamic Shuttling of AGO2 to the Nucleus Relieves MicroRNA Repression

Abstract

The Argonaute (AGO) and the Trinucleotide Repeat Containing 6 (TNRC6) family proteins are the core components of the mammalian microRNA-induced silencing complex (miRISC). This machinery mediates post-transcriptional silencing of mRNA targets by sequence-specific hybridization with microRNAs (miRNAs) in the cytoplasm. Localization to subcellular compartments is critical to understanding miRNA action, and our lab and others have shown that miRISC complexes are also present and active in mammalian nuclei. miRISC has several proposed nuclear functions, yet the biological significance and regulatory mechanisms of action of nuclear miRISC in mammalian cancer cells remain poorly understood. We observed that three different model systems that mimic colon tumor microenvironments: (1) 2D colorectal cancer cell, HCT116, cultures grown beyond a monolayer to high cell density, (2) 3D HCT116 tumor spheroid cultures grown in collagen matrix, and (3) primary tissue samples from malignant colon tumor and normal adjacent colon tissue, show significant nuclear enrichment of core RISC effector protein, AGO2. We tested the consequences of nuclear AGO2 localization on global microRNA regulation using a multipronged transcriptomics approach. We combined AGO2-eCLIP-sequencing, small-RNA-sequencing, and whole transcriptome sequencing to identify candidate gene targets bound by AGO2 that may be regulated by the most abundant microRNA families. We observed that the majority of AGO2-bound cytoplasmic 3'UTR miRISC targets were significantly upregulated, potentially de-repressed, when AGO2 is nuclear-enriched. Using an AGO2 construct with a nuclear localization sequence (NLS-AGO2-GFP), we were able to phenocopy the de-repression of cytoplasmic candidate genes and observe a correlation with a higher rate of migration. Our findings reveal dynamic relocalization of AGO2 to the nucleus in colon cancer culture systems and primary colon tumor tissue, which may impact our understanding of spatial regulation of microRNA action and reveal therapeutic targets that contribute to colon tumor progression. Another study from my thesis research provides a systemic evaluation of the effects of depleting microRNA biogenesis enzyme, DROSHA, and microRNA effector proteins, AGO1-4 and TNRC6A-C, on microRNA expression. Our findings help to define the boundaries of RNAi in a widely used cell line, HCT116, by refining a subset of abundant microRNAs that are DROSHA-dependent, associated with Argonaute proteins, and most likely to carry out robust gene regulation