The Hippo pathway effectors TAZ/YAP regulate dicer expression and microRNA biogenesis through Let-7

Abstract

MicroRNAs (miRNAs) are genome-encoded small double-stranded RNAs that have emerged as key regulators of gene expression and are implicated in most aspects of human development and disease. Canonical miRNA biogenesis involves processing of ∼70-nucleotide pre-miRNA hairpins by Dicer to generate mature ∼22-nucleotide miRNAs, which target complementary RNA sequences. Despite the importance of miRNA biogenesis, signaling mechanisms controlling this process are poorly defined. Here we demonstrate that the post-transcriptional regulation of Dicer is controlled by the cell density-mediated localization of the Hippo pathway effectors TAZ (transcriptional co-activator with PDZ-binding motif) and YAP (Yes-associated protein) (TAZ/YAP). We show that nuclear TAZ/YAP, which are abundant at low cell density, are required for efficient pre-miRNA processing. Knockdown of TAZ/YAP in low density cells, or density-mediated sequestration of TAZ/YAP into the cytoplasm, results in the defective processing of pre-miRNAs. Strikingly, one exception is Let-7, which accumulates upon loss of nuclear TAZ/YAP, leading to Let-7-dependent reduction in Dicer levels. Accordingly, inhibition of Let-7 rescues the miRNA biogenesis defects observed following TAZ/YAP knockdown. Thus, density-regulated TAZ/YAP localization defines a critical and previously unrecognized mechanism by which cells relay cell contact-induced cues to control miRNA biogenesis.

Keywords: Cell adhesion; Dicer; Hippo Pathway; Let-7; MicroRNA; RNA Processing; Signal Transduction; TAZ/YAP.

FIGURE 1.

Cell density-mediated TAZ/YAP localization controls miRNA processing. A, the levels of indicated pre-miRNAs and mature miRNAs were examined by Northern blotting in lysates isolated from MCF10A cells grown at low (L) and high (H) density. The relative levels of mature and pre-miRNAs were quantitated, and the ratios of mature/pre-miRNAs are shown as the average of three independent experiments ± S.D. (p < 0.05). U6 was probed as a loading control. LD, low density; HD, high density. B, MCF10A cells were grown at low and high cell density, and TAZ and YAP localization was examined by immunofluorescence confocal microscopy. Nuclei were stained with DAPI. C and D, MCF10A cells transfected with control siRNA, siRNA targeting TAZ/YAP (siT/Y), or siRNA targeting LATS1/2 (siL1/2 in D) were lysed and examined by Northern blotting for levels of the indicated pre-miRNA and mature miRNA. U6 was probed as a loading control. The relative levels of mature and pre-miRNAs from three independent experiments were quantitated, and the ratios of mature/pre-miRNAs are shown as the average ± S.E. (p < 0.05). The ratios of mature miRNAs/U6 from three independent experiments are also shown as the average ± S.E.

FIGURE 2.

TAZ/YAP control miRNA biogenesis by mediating Dicer stability. A, HEK-293 cells expressing the indicated pri-miRNA clusters were co-transfected with control siRNA (siCTL), siRNA targeting TAZ (siTAZ) or YAP (siYAP), or siRNA targeting both TAZ and YAP (siT/Y). Cell lysates were then examined for miRNA processing efficiency by Northern blotting for the levels of pre-miRNA and mature miRNA. The data from all the miRNAs from each cluster was quantitated, and the combined average ± S.E. of all experiments is shown in the right panel. U6 was probed as a loading control. B, MCF10A cells were transfected with siCTL or siT/Y, and then either total cell lysates or lysates from nuclear fractions were examined for pre-miRNAs. C, MCF10A cells were transfected with siCTL or siRNA targeting Dicer (siDicer), and the lysates were examined by Northern blotting for the levels of pre-miR-23a and miR-23a. U6 was probed as a loading control. D, MCF10A cells were transfected with siCTL or siT/Y, and the levels of Dicer protein were then examined by immunoblotting. TAZ and YAP, as well as GAPDH levels, were analyzed as controls. E, MCF10A cells were grown at low (LD) and high (HD) density, and the levels of Dicer, phospho-Ser-127-YAP (p-YAP), total YAP, and GAPDH were examined by immunoblotting. F, MCF10A cells were transfected with siCTL or siT/Y, and mRNA levels of the indicated genes were quantitated by qPCR. The data represents the average of three experiments ± S.E.

FIGURE 3.

TAZ/YAP govern the LIN28/Let-7 axis. A, MCF10A cells were co-transfected with the indicated siRNA and a control inhibitor (CTL inh) or Let-7b microRNA inhibitor (Let-7 inh), and cell lysates were subsequently examined by Northern blotting for the indicated pre-miRNAs and mature miRNAs. The relative expression of mature miRNA to pre-miRNA was quantified, comparing control siRNA (siCTL) with siRNA targeting TAZ/YAP (siT/Y) in the absence or presence of a Let-7 inhibitor. Experiments were performed three times independently, and the average ± S.E. is shown. B, MCF10A cells were transfected with siCTL or siT/Y, and the levels of pri-Let7a∼Let-7b were quantitated by real time qPCR. C, HEK293 cells were transfected with siCTL or siT/Y along with either a 1-kb or 1.5-kb Let-7 promoter-luciferase reporter, previously used to assess Let-7 transcriptional regulation (13). Relative normalized luciferase activity from the indicated cell lysates is shown. D, nuclear TAZ-4SA or YAP-5SA was co-expressed in HEK293 cells with the 1.5-kb Let-7 promoter-luciferase reporter, and the relative luciferase activity was examined. E, MCF10A cells transfected with siCTL or siT/Y were lysed and examined for the indicated proteins by immunoblotting. F, MCF10A cells were transfected with siCTL or siT/Y, and the mRNA levels of LIN28B were quantitated by real time qPCR. G, model for how cell density-mediated TAZ/YAP localization controls miRNA biogenesis.