The microRNAs of the miR‐200 family maintain the central characteristics of epithelia and inhibit tumor cell motility and invasiveness. Using the Ago‐HITS‐CLIP technology for transcriptome‐wide identification of direct microRNA targets in living cells, along with extensive validation to verify the reliability of the approach, we have identified hundreds of miR‐200a and miR‐200b targets, providing insights into general features of miRNA target site selection. Gene ontology analysis revealed a predominant effect of miR‐200 targets in widespread coordinate control of actin cytoskeleton dynamics. Functional characterization of the miR‐200 targets indicates that they constitute subnetworks that underlie the ability of cancer cells to migrate and invade, including coordinate effects on Rho‐ROCK signaling, invadopodia formation, MMP activity, and focal adhesions. Thus, the miR‐200 family maintains the central characteristics of the epithelial phenotype by acting on numerous targets at multiple levels, encompassing both cytoskeletal effectors that control actin filament organization and dynamics, and upstream signals that locally regulate the cytoskeleton to maintain cell morphology and prevent cell migration. ![][1] miR‐200 microRNAs are involved in the maintenance of epithelial integrity. Direct, transcriptome‐wide target detection and validation identifies genes functionally grouped as regulators of Rho GTPase signaling, invadopodia formation, metalloprotease activity and cell adhesion, which together regulate cell motility, migration and cancer metastasis. The EMBO Journal (2014) 33: 2040–2056 [1]: pending:yes

Members of the miR‐200 family are critical gatekeepers of the epithelial state, restraining expression of pro‐mesenchymal genes that drive epithelial–mesenchymal transition (EMT) and contribute to metastatic cancer progression. Here, we show that miR‐200c and another epithelial‐enriched miRNA, miR‐375, exert widespread control of alternative splicing in cancer cells by suppressing the RNA‐binding protein Quaking (QKI). During EMT, QKI‐5 directly binds to and regulates hundreds of alternative splicing targets and exerts pleiotropic effects, such as increasing cell migration and invasion and restraining tumour growth, without appreciably affecting mRNA levels. QKI‐5 is both necessary and sufficient to direct EMT‐associated alternative splicing changes, and this splicing signature is broadly conserved across many epithelial‐derived cancer types. Importantly, several actin cytoskeleton‐associated genes are directly targeted by both QKI and miR‐200c, revealing coordinated control of alternative splicing and mRNA abundance during EMT. These findings demonstrate the existence of a miR‐200/miR‐375/QKI axis that impacts cancer‐associated epithelial cell plasticity through widespread control of alternative splicing.

Deep-sequencing reveals extensive variation in the sequence of endogenously expressed microRNAs (termed ‘isomiRs’) in human cell lines and tissues, especially in relation to the 3′ end. From the immunoprecipitation of the microRNA-binding protein Argonaute and the sequencing of associated small RNAs, we observe extensive 3′-isomiR variation, including for miR-222 where the majority of endogenously expressed miR-222 is extended by 1–5 nt compared to the canonical sequence. We demonstrate this 3′ heterogeneity has dramatic implications for the phenotype of miR-222 transfected cells, with longer isoforms promoting apoptosis in a size (but not 3′ sequence)-dependent manner. The transfection of longer miR-222 isomiRs did not induce an interferon response, but did downregulate the expression of many components of the pro-survival PI3K-AKT pathway including PIK3R3, a regulatory subunit whose knockdown phenocopied the expression of longer 222 isoforms in terms of apoptosis and the inhibition of other PI3K-AKT genes. As this work demonstrates the capacity for 3′ isomiRs to mediate differential functions, we contend more attention needs to be given to 3′ variance given the prevalence of this class of isomiR.