Maturation of microRNA is hormonally regulated by a nuclear receptor.

Steroid hormones and their cognate nuclear receptors exert a wide spectrum of biological actions through regulation of transcriptional and posttranscriptional processes. However, the underlying molecular mechanism by which steroid hormones control posttranscriptional processes is largely unknown. We now report that estrogen receptor alpha (ERalpha) inhibits the maturation of a particular microRNA (miRNA) and thereby stabilizes the mRNA of an ERalpha target gene through the 3'UTR. Estrogen-bound ERalpha downregulated expression of a set of miRNAs in both animals and cultured cells. Activated ERalpha attenuated the processing of primary miRNAs into pre-miRNAs through estrogen-dependent association with the Drosha complex, resulting in stabilization of the transcript of an ERalpha target gene through its 3'UTR. Thus, a steroid hormone achieves posttranscriptional control by regulating the maturation of miRNA.

DEAD-box RNA helicase subunits of the Drosha complex are required for processing of rRNA and a subset of microRNAs.

MicroRNAs (miRNAs) control cell proliferation, differentiation and fate through modulation of gene expression by partially base-pairing with target mRNA sequences. Drosha is an RNase III enzyme that is the catalytic subunit of a large complex that cleaves pri-miRNAs with distinct structures into pre-miRNAs. Here, we show that both the p68 and p72 DEAD-box RNA helicase subunits in the mouse Drosha complex are indispensable for survival in mice, and both are required for primary miRNA and rRNA processing. Gene disruption of either p68 or p72 in mice resulted in early lethality, and in both p68(-/-) and p72(-/-) embryos, expression levels of a set of, but not all, miRNAs and 5.8S rRNA were significantly lowered. In p72(-/-) MEF cells, expression of p72, but not a mutant lacking ATPase activity, restored the impaired expression of miRNAs and 5.8S rRNA. Furthermore, we purified the large complex of mouse Drosha and showed it could generate pre-miRNA and 5.8S rRNA in vitro. Thus, we suggest that DEAD-box RNA helicase subunits are required for recognition of a subset of primary miRNAs in mDrosha-mediated processing.

Sepantronium bromide (YM155) induces disruption of the ILF3/p54(nrb) complex, which is required for survivin expression.

YM155, a small-molecule survivin suppressant, specifically binds to the transcription factor ILF3, which regulates the expression of survivin[1]. In this experiment we have demonstrated that p54(nrb) binds to the survivin promoter and regulates survivin expression. p54(nrb) forms a complex with ILF3, which directly binds to YM155. YM155 induces disruption of the ILF3/p54(nrb) complex, which results in a different subcellular localization between ILF3 and p54(nrb). Thus, identification of molecular targets of YM155 in suppression of the survivin pathway, might lead to development of its use as a novel potential target in cancers.

Combination of MS protein identification and bioassay of chromatographic fractions to identify biologically active substances from complex protein sources.

Purification of biologically active proteins from complex biological sources is a difficult task, usually requiring large amounts of sample and many separation steps. We found an active substance in a serum response element-dependent luciferase reporter gene bioassay in interstitial cystitis urine that we attempted to purify with column chromatography and the bioassay. With anion-exchange Mono Q and C4 reversed-phase columns, apparently sharp active peaks were obtained. However, more than 20 kinds of proteins were identified from the active fractions with MS, indicating that the purification was not complete. As further purification was difficult, we chose a candidate molecule by means of studying the correlation between MS protein identification scores and bioassay responses of chromatographic fractions near the active peaks. As a result, epidermal growth factor (EGF) was nominated as a candidate molecule among the identified proteins because the elution profile of EGF was consistent with that of the bioassay, and the correlation coefficient of EGF between MS protein identification scores and bioassay responses was the highest among all the identified proteins. With recombinant EGF and anti-EGF and anti-EGF receptor antibodies, EGF was confirmed to be the desired substance in interstitial cystitis urine. This approach required only 20 ml of urine sample and two column chromatographic steps. The combination of MS protein identification and bioassay of chromatographic fractions may be useful for identifying biologically active substances from complex protein sources.

Inhibition of c-Rel DNA binding is critical for the anti-inflammatory effects of novel PIKfyve inhibitor.

Aberrant production of proinflammatory cytokines is linked to many autoimmune diseases, and their inhibition by small molecule compounds is considered beneficial. Here, we performed phenotypic screening in IFNγ/LPS-activated RAW264.7, mouse macrophage cells, and discovered AS2677131 and AS2795440 as novel and potent inhibitors of IL-12p40, a subunit of IL-23. Interestingly, these compounds exhibited unique pharmacological activities in their inhibition of the production of IL-12p40, IL-6 and IL-1ß but not TNFα in activated macrophages or dendritic cells, and expression of IgM-induced MHC class II on B cells. To reveal these mechanisms, we synthesized two different activity probes which were structurally related to the AS compounds, and identified probe-specific binding proteins, including PIKfyve, a Class III PI kinase. The AS compounds inhibited PIKfyve activity and mimicked the properties of PIKfyve-deficient cells, eventually validating PIKfyve as target molecule. Regarding mechanism, AS2677131 regulated DNA binding activity of c-Rel on IL-12p40 and IL-1ß promoter. As expected, a PIKfyve inhibitor prevented the development of arthritis in rats. Taken together, our findings of the novel and potent PIKfyve inhibitors AS2677131 and AS2795440 reveal the critical role of PIKfyve in proinflammatory cytokine production and B cell activation, and may indicate a potential new therapeutic option for treatment of inflammatory diseases.

RMI1 deficiency in mice protects from diet and genetic-induced obesity.

The aim of this study is to discover and characterize novel energy homeostasis-related molecules. We screened stock mouse embryonic stem cells established using the exchangeable gene trap method, and examined the effects of deficiency of the target gene on diet and genetic-induced obesity. The mutant strain 0283, which has an insertion at the recQ-mediated genome instability 1 (RMI1) locus, possesses a number of striking features that allow it to resist metabolic abnormalities. Reduced RMI1 expression, lower fasting-blood glucose and a reduced body weight (normal diet) were observed in the mutant mice. When fed a high-fat diet, the mutant mice were resistant to obesity, and also showed improved glucose intolerance and reduced abdominal fat tissue mass and food intake. In addition, the mutants were also resistant to obesity induced by the lethal yellow agouti (A(y)) gene. Endogenous RMI1 genes were found to be up-regulated in the liver and adipose tissue of KK-A(y) mice. RMI1 is a component of the Bloom's syndrome gene helicase complex that maintains genome integrity and activates cell-cycle checkpoint machinery. Interestingly, diet-induced expression of E2F8 mRNA, which is an important cell cycle-related molecule, was suppressed in the mutant mice. These results suggest that the regulation of energy balance by RMI1 is attributable to the regulation of food intake and E2F8 expression in adipose tissue. Taken together, these findings demonstrate that RMI1 is a novel molecule that regulates energy homeostasis.

Absence of increased alpha1-microglobulin in IgA nephropathy proteinuria.

To search for biomarkers of IgA nephropathy, protein profiles of urine samples from patients with IgA nephropathy and normal volunteers were compared using two-dimensional DIGE. Most of the 172 spots identified in the urine were serum proteins, and their amounts in IgA nephropathy urine were much higher than those in normal urine; this can be explained as proteinuria caused by glomerular dysfunction. However, only alpha(1)-microglobulin, also one of the major serum proteins, in IgA nephropathy urine was not higher in amount than that in normal urine. We confirmed using ELISA analysis that the amounts of transferrin and albumin in IgA nephropathy and diabetic nephropathy urine were much higher than those in normal urine, whereas the amount of alpha(1)-microglobulin in IgA nephropathy urine was not higher than that in normal urine and was much lower than that in diabetic nephropathy urine. Approximately 50% of alpha(1)-microglobulin forms a complex with IgA in serum. These results suggest that alpha(1)-microglobulin in IgA nephropathy urine is a characteristic protein and might be a biomarker for IgA nephropathy and that alpha(1)-microglobulin might have a relationship with IgA nephropathy pathology.