The positive regulatory machinery in the microRNA (miRNA) processing pathway is relatively well characterized, but negative regulation of the pathway is largely unknown. Here we show that a complex of nuclear factor 90 (NF90) and NF45 proteins functions as a negative regulator in miRNA biogenesis. Primary miRNA (pri-miRNA) processing into precursor miRNA (pre-miRNA) was inhibited by overexpression of the NF90 and NF45 proteins, and considerable amounts of pri-miRNAs accumulated in cells coexpressing NF90 and NF45. Treatment of cells overexpressing NF90 and NF45 with an RNA polymerase II inhibitor, ␣-amanitin, did not reduce the amounts of pri-miRNAs, suggesting that the accumulation of pri-miRNAs is not due to transcriptional activation. In addition, the NF90 and NF45 complex was not found to interact with the Microprocessor complex, which is a processing factor of primiRNAs, but was found to bind endogenous pri-miRNAs. NF90-NF45 exhibited higher binding activity for pri-let-7a than pri-miR-21. Of note, depletion of NF90 caused a reduction of pri-let-7a and an increase of mature let-7a miRNA, which has a potent antiproliferative activity, and caused growth suppression of transformed cells. These findings suggest that the association of the NF90-NF45 complex with pri-miRNAs impairs access of the Microprocessor complex to the pri-miRNAs, resulting in a reduction of mature miRNA production.MicroRNAs (miRNAs) constitute a class of noncoding small RNAs that function as repressors for eukaryotic gene regulation by binding to the 3Ј untranslated regions of target mRNAs (2). This binding causes mRNA cleavage or translational inhibition of the mRNA, depending upon the degree of complementarity. The lengths of miRNAs are 21 to 23 nucleotides (nt), and over 500 miRNAs have been discovered in mammals. miRNAs regulate the expression of a large number of genes (38) that are involved in cell proliferation, apoptosis, hematopoietic differentiation, viral infection, and tumorigenesis (4,5,7,22,26,32,39,45).In mammals, miRNA genes are transcribed by RNA polymerase II as primary miRNAs (pri-miRNAs) (36). These primiRNAs are processed into precursor miRNAs (pre-miRNAs) by the Microprocessor complex (8,13,20,31,33). Another complex comprised of exportin-5 and RanGTP transports the pre-miRNAs from the nucleus to the cytoplasm (3, 40, 58). In the cytoplasm, Dicer, a cytoplasmic RNase III enzyme, cleaves the pre-miRNAs to approximate 22-nt mature miRNA duplexes with 2-nt 3Ј overhangs (14,24,28). One strand of the duplex is incorporated into the RNA-induced silencing complex (12,19,29,41,51). The single strand of RNA guides the RNA-induced silencing complex to the target mRNA with sequence complementarity, which leads either to mRNA cleavage or to translational repression (12,24,41,44).The Microprocessor complex, which cleaves pri-miRNA to pre-miRNA during miRNA biogenesis, is comprised of a nuclear RNase III enzyme, Drosha, and its cofactor, DGCR8 (8,13,20). In addition to the Microprocessor complex, excessively expressed Drosha forms ...

MicroRNA-7 (miR-7)has been characterized as an anti-oncogenic microRNA (miRNA) in several cancers, including hepatocellular carcinoma (HCC). However, the mechanism for the regulation of miR-7 production in tumors remains unclear. Here, we identified nuclear factor 90 (NF90) and NF45 complex (NF90-NF45) as negative regulators of miR-7 processing in HCC. Expression of NF90 and NF45 was significantly elevated in primary HCC tissues compared with adjacent non-tumor tissues. To examine which miRNAs are controlled by NF90-NF45, we performed an miRNA microarray and quantitative RT-PCR analyses of HCC cell lines. Depletion of NF90 resulted in elevated levels of mature miR-7, whereas the expression of primary miR-7-1 (pri-miR-7-1) was decreased in cells following knockdown of NF90. Conversely, the levels of mature miR-7 were reduced in cells overexpressing NF90 and NF45, although pri-miR-7-1 was accumulated in the same cells. Furthermore, NF90-NF45 was found to bind pri-miR-7-1 in vitro These results suggest that NF90-NF45 inhibits the pri-miR-7-1 processing step through the binding of NF90-NF45 to pri-miR-7-1. We also found that levels of the EGF receptor, an oncogenic factor that is a direct target of miR-7, and phosphorylation of AKT were significantly decreased in HCC cell lines depleted of NF90 or NF45. Of note, knockdown of NF90 or NF45 caused a reduction in the proliferation rate of HCC cells. Taken together, NF90-NF45 stimulates an elevation of EGF receptor levels via the suppression of miR-7 biogenesis, resulting in the promotion of cell proliferation in HCC.

Reduced expression of DICER, a key enzyme in the miRNA pathway, is frequently associated with aggressive, invasive disease and poor survival in various malignancies. Regulation of DICER expression is, however, poorly understood. Here, we show that NF90/NF110 facilitates DICER expression by controlling the processing of a miRNA, miR-3173, that is embedded in DICER pre-mRNA. Since miR-3173 in turn targets NF90, a feedback amplification loop controlling DICER expression is established. In a nude mouse model, NF90 overexpression reduced proliferation of ovarian cancer cells and significantly reduced tumor size and metastasis while overexpression of miR-3173 dramatically increased metastasis in a NF90- and DICER-dependent manner. Clinically, low NF90 expression and high miR-3173-3p expression were found to be independent prognostic markers of poor survival in a cohort of ovarian carcinoma patients. These findings suggest that, by facilitating DICER expression, NF90 can act as a suppressor of ovarian carcinoma.

d MicroRNAs (miRNAs) are involved in the progression and suppression of various diseases through translational inhibition of target mRNAs. Therefore, the alteration of miRNA biogenesis induces several diseases. The nuclear factor 90 (NF90)-NF45 complex is known as a negative regulator in miRNA biogenesis. Here, we showed that NF90-NF45 double-transgenic (dbTg) mice develop skeletal muscle atrophy and centronuclear muscle fibers in adulthood. Subsequently, we found that the levels of myogenic miRNAs, including miRNA 133a (miR-133a), which promote muscle maturation, were significantly decreased in the skeletal muscle of NF90-NF45 dbTg mice compared with those in wild-type mice. However, levels of primary transcripts of the miRNAs (pri-miRNAs) were clearly elevated in NF90-NF45 dbTg mice. This result indicated that the NF90-NF45 complex suppressed miRNA production through inhibition of pri-miRNA processing. This finding was supported by the fact that processing of pri-miRNA 133a-1 (primiR-133a-1) was inhibited via binding of NF90-NF45 to the pri-miRNA. Finally, the level of dynamin 2, a causative gene of centronuclear myopathy and concomitantly a target of miR-133a, was elevated in the skeletal muscle of NF90-NF45 dbTg mice. Taken together, we conclude that the NF90-NF45 complex induces centronuclear myopathy through increased dynamin 2 expression by an NF90-NF45-induced reduction of miR-133a expression in vivo.M icroRNAs (miRNAs) are functional small noncoding RNAs 21 to 23 nucleotides in length. miRNAs bind to 3= untranslated regions (UTRs) of target mRNAs, leading to either mRNA degradation or translational inhibition. The functions of miRNAs influence biological phenomena and diseases such as development (1, 2), cell proliferation (3), differentiation (4), apoptosis (5), as well as tumorigenesis (6, 7).The biogenesis of miRNAs involves several steps (8). First, miRNA genes are transcribed as primary miRNAs (pri-miRNAs) by RNA polymerase II (9). Subsequently, these pri-miRNAs are processed into precursor miRNAs (pre-miRNAs) by a microprocessor complex composed of Drosha and DGCR8 (10-14). Thereafter, the pre-miRNAs are transported from the nucleus to the cytoplasm (15-17) and processed into mature miRNA duplexes by the Dicer complex (18)(19)(20). The functional strand of the duplex (mature miRNA) is loaded into the RNA-induced silencing complex (RISC) (21-23). As part of the RISC, miRNA binds to target mRNAs and induces their translational inhibition or degradation (20,(22)(23)(24). Recently, it was reported that some RNA-binding proteins negatively regulate miRNA biogenesis. For example, Lin28A/B (25), the Musashi homolog 2/Hu antigen R complex (26), and the nuclear factor 90 (NF90; also referred to as interleukin enhancer binding factor 3 [ILF3], NFAR1, or DRBP76)-nuclear factor 45 (NF45) complex (27) suppress miRNA processing through binding to pri-or pre-miRNAs.NF90 contains a functional nuclear localization signal, two double-stranded RNA (dsRNA)-binding motifs, a zinc finger nucleic acid-binding domain, an...

Abstract MicroRNAs (miRNAs) are predicted to regulate the expression of >60% of mammalian genes and play fundamental roles in most biological processes. Deregulation of miRNA expression is a hallmark of most cancers and further investigation of mechanisms controlling miRNA biogenesis is needed. The double stranded RNA-binding protein, NF90 has been shown to act as a competitor of Microprocessor for a limited number of primary miRNAs (pri-miRNAs). Here, we show that NF90 has a more widespread effect on pri-miRNA biogenesis than previously thought. Genome-wide approaches revealed that NF90 is associated with the stem region of 38 pri-miRNAs, in a manner that is largely exclusive of Microprocessor. Following loss of NF90, 22 NF90-bound pri-miRNAs showed increased abundance of mature miRNA products. NF90-targeted pri-miRNAs are highly stable, having a lower free energy and fewer mismatches compared to all pri-miRNAs. Mutations leading to less stable structures reduced NF90 binding while increasing pri-miRNA stability led to acquisition of NF90 association, as determined by RNA electrophoretic mobility shift assay (EMSA). NF90-bound and downregulated pri-miRNAs are embedded in introns of host genes and expression of several host genes is concomitantly reduced. These data suggest that NF90 controls the processing of a subset of highly stable, intronic miRNAs.

Expression changes in three miRNAs could be detected in the eye before histologic EAU. Kinetic changes of these miRNAs in the eye paralleled those of IL-17. The possibility that miRNAs can affect IL-17 suggests that miRNAs in the retina regulate the development of EAU.

BackgroundObesity-induced liver disease (nonalcoholic fatty liver disease, NAFLD) is now the commonest cause of chronic liver disease in affluent nations. There are presently no proven treatments for NAFLD or its more severe stage, nonalcoholic steatohepatitis (NASH). Bofutsushosan (BTS), a Japanese herbal (Kampo) medicine, long used as an anti-obesity medicine in Japan and other Asian countries, has been shown to reduce body weight and improve insulin resistance (IR) and hepatic steatosis. The precise mechanism of action of BTS, however, remains unclear. To evaluate the ability of BTS to prevent the development of NASH, and determine the mediators and pathways involved.MethodsC57BL/6 mice were injected intra-peritoneally with gold-thioglucose and fed a high-fat diet (HF) or HF diet admixed with either 2 or 5 % BTS for 12 weeks. The effectiveness of BTS in attenuating features of NASH and the mechanisms through which BTS attenuated NASH were then assayed through an assessment of the anthropometric, radiological, biochemical and histological parameters.ResultsBTS attenuated the progression of NASH through induction of adiponectin and its receptors along with an induction of PPAR-α and PPAR-γ, decreased expression of SREBP-1c, increased hepatic fatty acid oxidation and increased hepatic export of triglycerides. BTS moreover, reduced IR through phosphorylation of the protein kinase, Akt.ConclusionsBTS through induction of adiponectin signaling and Akt attenuated development of NASH. Identification of the active entity in BTS should allow development of novel treatments for NASH.Electronic supplementary materialThe online version of this article (doi:10.1007/s00535-013-0852-8) contains supplementary material, which is available to authorized users.

Through screening of a murine brain cDNA library, we have isolated two brain specific cDNAs encoding highly homologous proteins, named 921-L and 921-S, comprised of 134 amino acids with 80% identity. Immunohistological study with the mAbs raised against the bacterially expressed 921 proteins showed that 921-L protein is distributed at the dendritic region and 921-S at the neuronal somatic surface. Immuno-electron microscopic study revealed that both 921 proteins are localized at the presynaptic terminal, indicating that the 921 proteins are differentially expressed at the dendritic and somatic presynapses.Through screening of a murine brain cDNA library with the repetitive DNA as a probe, we have isolated a brain specific cDNA (921-L) containing (GGA)r~(GAA)n repeat and another cDNA (92 l-S) homologous to 921-L. Both cDNAs encode two highly homologous proteins which are expressed at the presynaptic terminal. Their distributions are distinct: 921-L protein is expressed at the dendritic synapse and 921-S at the somatic synapse. In this report we present these findings including the cDNA isolation process.