Abstract
BackgroundThe RNA-binding protein Argonaute 2 (AGO2) is a key effector of RNA-silencing pathways It exerts a pivotal role in microRNA maturation and activity and can modulate chromatin remodeling, transcriptional gene regulation and RNA splicing. Estrogen receptor beta (ERβ) is endowed with oncosuppressive activities, antagonizing hormone-induced carcinogenesis and inhibiting growth and oncogenic functions in luminal-like breast cancers (BCs), where its expression correlates with a better prognosis of the disease.ResultsApplying interaction proteomics coupled to mass spectrometry to characterize nuclear factors cooperating with ERβ in gene regulation, we identify AGO2 as a novel partner of ERβ in human BC cells. ERβ–AGO2 association was confirmed in vitro and in vivo in both the nucleus and cytoplasm and is shown to be RNA-mediated. ChIP-Seq demonstrates AGO2 association with a large number of ERβ binding sites, and total and nascent RNA-Seq in ERβ + vs ERβ − cells, and before and after AGO2 knock-down in ERβ + cells, reveals a widespread involvement of this factor in ERβ-mediated regulation of gene transcription rate and RNA splicing. Moreover, isolation and sequencing by RIP-Seq of ERβ-associated long and small RNAs in the cytoplasm suggests involvement of the nuclear receptor in RISC loading, indicating that it may also be able to directly control mRNA translation efficiency and stability.ConclusionsThese results demonstrate that AGO2 can act as a pleiotropic functional partner of ERβ, indicating that both factors are endowed with multiple roles in the control of key cellular functions.
Highlights
The RNA-binding protein Argonaute 2 (AGO2) is a key effector of RNA-silencing pathways It exerts a pivotal role in microRNA maturation and activity and can modulate chromatin remodeling, transcriptional gene regulation and RNA splicing
We previously showed that stable expression of estrogen receptor β (ERβ) fused to a TAP tag at either the C-terminus (Ct-ERβ) or N-terminus (Nt-ERβ), suitable for proteomics analyses, causes growth inhibition and re-programming of miRNA expression and the cell proteome in human luminal-like MCF-7 breast cancers (BCs) cells [25], in line with results obtained in other laboratories [27,28,29]
Evaluation of the functional significance of the gene expression changes detected in ERβ-expressing cells, performed by IPA comparative analysis, revealed that all the top ten functional annotations identified relate to key cancer cell characteristics, including regulation of cellular movement, cell-to-cell signaling and interactions, cell morphology, growth and proliferation, Fig. 1 Effects of unliganded ERβ on the BC cell transcriptome and alternative RNA splicing. a The fraction of differentially expressed genes detected in both Ct-ERβ- and Nt-ERβ-expressing cells or in only one of the two cell lines. b Functional annotation by Ingenuity Comparative Analysis on genes differentially expressed in BC cells expressing either Ct-ERβ or Nt-ERβ (fold change cut-off |1.5|, false discovery rate (FDR) ≤ 0.05)
Summary
The RNA-binding protein Argonaute 2 (AGO2) is a key effector of RNA-silencing pathways It exerts a pivotal role in microRNA maturation and activity and can modulate chromatin remodeling, transcriptional gene regulation and RNA splicing. ERβ shows additive effects with anti-estrogens in promotion of apoptotic cell death and cell cycle inhibition [19, 20], and for this reason has been proposed as a marker of tumor responsiveness to endocrine therapy [21, 22] This receptor can bind estrogenic compounds, thereby exerting a modulatory role on the functions of the oncogenic ERα, the other estrogen receptor subtype active in cancer cells, by dimerizing with it and thereby modifying its activity on target genes [16, 23], in the absence of ligand it exhibits significant effects in BC cells [24], including, among others, miRNA-mediated post-transcriptional regulation of the BC cell proteome [25]. The presence of unliganded ERβ is a typical condition during specific phases of the menstrual cycle, before puberty, and in post-menopausal women, when this receptor might compensate for the absence of circulating hormones with regard to cell functions
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