Hiroshi I. Suzuki

Modulation of microRNA processing by p53

MicroRNAs (miRNAs) have emerged as key post-transcriptional regulators of gene expression, involved in diverse physiological and pathological processes. Although miRNAs can function as both tumour suppressors and oncogenes in tumour development, a widespread downregulation of miRNAs is commonly observed in human cancers and promotes cellular transformation and tumorigenesis. This indicates an inherent significance of small RNAs in tumour suppression. However, the connection between tumour suppressor networks and miRNA biogenesis machineries has not been investigated in depth. Here we show that a central tumour suppressor, p53, enhances the post-transcriptional maturation of several miRNAs with growth-suppressive function, including miR-16-1, miR-143 and miR-145, in response to DNA damage. In HCT116 cells and human diploid fibroblasts, p53 interacts with the Drosha processing complex through the association with DEAD-box RNA helicase p68 (also known as DDX5) and facilitates the processing of primary miRNAs to precursor miRNAs. We also found that transcriptionally inactive p53 mutants interfere with a functional assembly between Drosha complex and p68, leading to attenuation of miRNA processing activity. These findings suggest that transcription-independent modulation of miRNA biogenesis is intrinsically embedded in a tumour suppressive program governed by p53. Our study reveals a previously unrecognized function of p53 in miRNA processing, which may underlie key aspects of cancer biology.

Activin-Nodal signaling is involved in propagation of mouse embryonic stem cells

Embryonic stem (ES) cells are self-renewing cells that maintain pluripotency to differentiate into all types of cells. Because of their potential to provide a variety of tissues for use in regenerative medicine, there is great interest in the identification of growth factors that govern these unique properties of ES cells. However, the signaling pathways controlling ES cell proliferation remain largely unknown. Since transforming growth factor β (TGFβ) superfamily members have been implicated in the processes of early embryogenesis, we investigated their roles in ES cell self-renewal. Inhibition of activin-Nodal-TGFβ signaling by Smad7 or SB-431542 dramatically decreased ES cell proliferation without decreasing ES pluripotency. By contrast, inhibition of bone morphogenetic protein (BMP) signaling by Smad6 did not exhibit such effects, suggesting that activin-Nodal-TGFβ signaling, but not BMP signaling, is indispensable for ES cell propagation. In serum-free culture, supplementation of recombinant activin or Nodal, but not TGFβ or BMP, significantly enhanced ES cell propagation without affecting pluripotency. We also found that activin-Nodal signaling was constitutively activated in an autocrine fashion in serum-free cultured ES cells, and that inhibition of such endogenous signaling by SB-431542 decreased ES cell propagation in serum-free conditions. These findings suggest that endogenously activated autocrine loops of activin-Nodal signaling promote ES cell self-renewal.

MCPIP1 Ribonuclease Antagonizes Dicer and Terminates MicroRNA Biogenesis through Precursor MicroRNA Degradation

MicroRNAs (miRNAs) are versatile regulators of gene expression and undergo complex maturation processes. However, the mechanism(s) stabilizing or reducing these small RNAs remains poorly understood. Here we identify mammalian immune regulator MCPIP1 (Zc3h12a) ribonuclease as a broad suppressor of miRNA activity and biogenesis, which counteracts Dicer, a central ribonuclease in miRNA processing. MCPIP1 suppresses miRNA biosynthesis via cleavage of the terminal loops of precursor miRNAs (pre-miRNAs). MCPIP1 also carries a vertebrate-specific oligomerization domain important for pre-miRNA recognition, indicating its recent evolution. Furthermore, we observed potential antagonism between MCPIP1 and Dicer function in human cancer and found a regulatory role of MCPIP1 in the signaling axis comprising miR-155 and its target c-Maf. These results collectively suggest that the balance between processing and destroying ribonucleases modulates miRNA biogenesis and potentially affects pathological miRNA dysregulation. The presence of this abortive processing machinery and diversity of MCPIP1-related genes may imply a dynamic evolutional transition of the RNA silencing system.

Emerging complexity of microRNA generation cascades

MicroRNA (miRNA) modules are built in genetic networks as a complex regulatory layer directing post-transcriptional gene regulation. miRNAs coordinate a broad spectra of gene expression programs mainly through modulation of mRNA metabolism. Perturbations of miRNA networks are linked to a wide variety of pathological processes, including cardiovascular diseases and cancer. While the mechanisms regulating miRNA biogenesis were previously poorly understood, recent findings have shed light on the regulatory mechanisms of miRNAs themselves, especially their biogenesis. Multiple steps of miRNA maturation could potentially provide a variety of regulatory options to generate mature miRNAs differentially and produce gradation in miRNA processing efficiency. Several studies have demonstrated that miRNA maturation pathways crosstalk with intracellular signalling molecules, including p53, Smad proteins and estrogen receptor. Other lines of evidence have demonstrated the involvement of multiple RNA binding proteins in biased processing of different miRNA species. This review summarizes accumulating evidence for the emerging complexity and dynamics of regulated miRNA processing. These findings will lead to better understanding of miRNA dynamics in various pathogenetic pathways and provide the molecular basis for diagnostic and therapeutic strategies based on small RNA biology.

MicroRNA regulons in tumor microenvironment

Cancer initiation and progression are defined by the behavior of cancer cells per se and the development of tumor tissues, both of which are modulated by crosstalk between cancer cells and the surrounding microenvironment. Advances in cancer research have highlighted the significance of constant evolution of the tumor microenvironment, leading to tumor formation, metastasis and refractoriness to therapy. MicroRNAs (miRNAs) are small non-coding RNAs that function as major players of posttranscriptional gene regulation in diverse biological processes. They function as both tumor suppressors and promoters in many aspects of the autonomous behavior of cancer cells. Theoretically, dysfunction in the gene regulatory networks of cancer cells is one of the major driving forces for alterations of ostensibly normal surrounding cells. In this context, the core targets of miRNAs, termed miRNA regulons, are currently being expanded to include various modulators of the tumor microenvironment. Recent advances have highlighted two important roles played by miRNAs in the evolution of tumor microenvironments: miRNAs in tumor cells transform the microenvironment via non-cell-autonomous mechanisms, and miRNAs in neighboring cells stabilize cancer hallmark traits. These observations epitomize the distal and proximal functions of miRNAs in tumor microenvironments, respectively. Such regulation by miRNAs affects tumor angiogenesis, immune invasion and tumor–stromal interactions. This review summarizes recent findings on the mechanisms of miRNA-mediated regulation of tumor microenvironments, with a perspective on the design of therapeutic interventions.

Dynamics of microRNA biogenesis: crosstalk between p53 network and microRNA processing pathway

MicroRNAs (miRNAs) are pivotal regulators involved in various biological functions through the post-transcriptional regulation of gene expression. Alterations of miRNA expression and function contribute to both physiological and pathological processes such as development and cancer. While their roles have been attracting more attention in connection with tumor development, the mechanisms regulating miRNA biogenesis have not been well understood. Accumulating evidences have revealed the dynamic regulation of miRNA biosynthesis by several regulatory factors and demonstrated the complexity of miRNA-mediated gene regulation. In addition, several reports showed the interplay between the p53 tumor suppressor network and the miRNA-mediated gene regulatory system. We recently found that p53 modulates miRNA maturation at the processing step of primary miRNA transcripts, unraveling a novel function of p53. Here, we review the recent understanding of functional links between miRNA biogenesis and intracellular signaling pathways, with particular focus on the crosstalk between the p53 network and the miRNA biogenesis machinery. Further characterization of controlling elements for miRNA production and activity would provide important insights for a comprehensive understanding of the miRNA function in health and disease.

TGF-β-induced mesenchymal transition of MS-1 endothelial cells requires Smad-dependent cooperative activation of Rho signals and MRTF-A

Endothelial-mesenchymal transition (EndMT) plays important roles in various physiological and pathological processes. While signals mediated by transforming growth factor (TGF)-β have been implicated in EndMT, the molecular mechanisms underlying it remain to be fully elucidated. Here, we examined the effects of TGF-β signals on the EndMT of mouse pancreatic microvascular endothelial cells (MS-1). By addition of TGF-β2, MS-1 cells underwent mesenchymal transition characterized by re-organization of actin stress fibre and increased expression of various mesenchymal markers such as α-smooth muscle actin (α-SMA) through activation of Rho signals. Whereas activation of Rho signals via TGF-β-induced non-Smad signals has been implicated in epithelial-mesenchymal transition (EMT), we found that Arhgef5, a guanine nucleotide exchange factor, is induced by Smad signals and contributes to the TGF-β2-induced α-SMA expression in MS-1 cells. We also found that TGF-β2 induces the expression of myocardin-related transcription factor-A (MRTF-A) in a Smad-dependent fashion and its nuclear accumulation in MS-1 cells and that MRTF-A is required and sufficient for TGF-β2-induced α-SMA expression. These results indicate that activation of Smad signals by TGF-β2 have dual effects on the activation of Rho signals and MRTF-A leading to the mesenchymal transition of MS-1 endothelial cells.

Bone morphogenetic protein-9 inhibits lymphatic vessel formation via activin receptor-like kinase 1 during development and cancer progression

Significance Because lymphatic vessels (LVs) play critical roles not only in physiological processes such as maintenance of fluid homeostasis but also in pathological conditions including cancer metastasis, identification of factors that control LV formation is crucial. Signals mediated by activin receptor-like kinase 1 (ALK-1), a receptor for bone morphogenetic protein 9 (BMP-9), have been implicated in the formation of blood vessels because of its linkage to a human vascular disease. However, their roles in LV formation largely remain to be elucidated. Here, we show that BMP-9/ALK-1 signals inhibit LV formation in physiological and pathological conditions. Furthermore, we elucidated its molecular mechanisms in detail, using both in vivo and in vitro systems. These findings will help develop therapeutic strategies for LV-related diseases such as lymphedema and cancer. Lymphatic vessels (LVs) play critical roles in the maintenance of fluid homeostasis and in pathological conditions, including cancer metastasis. Although mutations in ALK1, a member of the transforming growth factor (TGF)-β/bone morphogenetic protein (BMP) receptor family, have been linked to hereditary hemorrhagic telangiectasia, a human vascular disease, the roles of activin receptor-like kinase 1 (ALK-1) signals in LV formation largely remain to be elucidated. We show that ALK-1 signals inhibit LV formation, and LVs were enlarged in multiple organs in Alk1-depleted mice. These inhibitory effects of ALK-1 signaling were mediated by BMP-9, which decreased the number of cultured lymphatic endothelial cells. Bmp9-deficient mouse embryos consistently exhibited enlarged dermal LVs. BMP-9 also inhibited LV formation during inflammation and tumorigenesis. BMP-9 downregulated the expression of the transcription factor prospero-related homeobox 1, which is necessary to maintain lymphatic endothelial cell identity. Furthermore, silencing prospero-related homeobox 1 expression inhibited lymphatic endothelial cell proliferation. Our findings reveal a unique molecular basis for the physiological and pathological roles of BMP-9/ALK-1 signals in LV formation.

Sjögren Syndrome Antigen B (SSB)/La Promotes Global MicroRNA Expression by Binding MicroRNA Precursors through Stem-Loop Recognition

Background: MicroRNA biogenesis is a multistep process regulated by RNA-binding proteins. Results: Sjögren syndrome antigen B (SSB)/La binds stem-loop precursor microRNAs (pre-miRNA) and is required for microRNA expression. Conclusion: La/SSB promotes global microRNA expression by stabilizing pre-miRNAs from nuclease-mediated decay. Significance: This study reveals a novel concept of pre-miRNA holding complex that protects and escorts pre-miRNAs in microRNA biogenesis pathway. MicroRNAs (miRNA) control numerous physiological and pathological processes. Typically, the primary miRNA (pri-miRNA) transcripts are processed by nuclear Drosha complex into ∼70-nucleotide stem-loop precursor miRNAs (pre-miRNA), which are further cleaved by cytoplasmic Dicer complex into ∼21-nucleotide mature miRNAs. However, it is unclear how nascent pre-miRNAs are protected from ribonucleases, such as MCPIP1, that degrade pre-miRNAs to abort miRNA production. Here, we identify Sjögren syndrome antigen B (SSB)/La as a pre-miRNA-binding protein that regulates miRNA processing in vitro. All three RNA-binding motifs (LAM, RRM1, and RRM2) of La/SSB are required for efficient pre-miRNA binding. Intriguingly, La/SSB recognizes the characteristic stem-loop structure of pre-miRNAs, of which the majority lack a 3′ UUU terminus. Moreover, La/SSB associates with endogenous pri-/pre-miRNAs and promotes miRNA biogenesis by stabilizing pre-miRNAs from nuclease (e.g. MCPIP1)-mediated decay in mammalian cells. Accordingly, we observed positive correlations between the expression status of La/SSB and Dicer in human cancer transcriptome and prognosis. These studies identify an important function of La/SSB as a global regulator of miRNA expression, and implicate stem-loop recognition as a major mechanism that mediates association between La/SSB and diverse RNA molecules.

Super-Enhancer-Mediated RNA Processing Revealed by Integrative MicroRNA Network Analysis

Super-enhancers are an emerging subclass of regulatory regions controlling cell identity and disease genes. However, their biological function and impact on miRNA networks are unclear. Here, we report that super-enhancers drive the biogenesis of master miRNAs crucial for cell identity by enhancing both transcription and Drosha/DGCR8-mediated primary miRNA (pri-miRNA) processing. Super-enhancers, together with broad H3K4me3 domains, shape a tissue-specific and evolutionarily conserved atlas of miRNA expression and function. CRISPR/Cas9 genomics revealed that super-enhancer constituents act cooperatively and facilitate Drosha/DGCR8 recruitment and pri-miRNA processing to boost cell-specific miRNA production. The BET-bromodomain inhibitor JQ1 preferentially inhibits super-enhancer-directed cotranscriptional pri-miRNA processing. Furthermore, super-enhancers are characterized by pervasive interaction with DGCR8/Drosha and DGCR8/Drosha-regulated mRNA stability control, suggesting unique RNA regulation at super-enhancers. Finally, super-enhancers mark multiple miRNAs associated with cancer hallmarks. This study presents principles underlying miRNA biology in health and disease and an unrecognized higher-order property of super-enhancers in RNA processing beyond transcription.