Blending and separating dynamics of RNA-binding proteins develop architectural splicing networks spreading throughout the nucleus.

The eukaryotic nucleus has a highly organized structure. Although the spatiotemporal arrangement of spliceosomes on nascent RNA drives splicing, the nuclear architecture that directly supports this process remains unclear. Here, we show that RNA-binding proteins (RBPs) assembled on RNA form meshworks in human and mouse cells. Core and accessory RBPs in RNA splicing make two distinct meshworks adjacently but distinctly distributed throughout the nucleus. This is achieved by mutual exclusion dynamics between the charged and uncharged intrinsically disordered regions (IDRs) of RBPs. These two types of meshworks compete for spatial occupancy on pre-mRNA to regulate splicing. Furthermore, the optogenetic enhancement of the RBP meshwork causes aberrant splicing, particularly of genes involved in neurodegeneration. Genetic mutations associated with neurodegenerative diseases are often found in the IDRs of RBPs, and cells harboring these mutations exhibit impaired meshwork formation. Our results uncovered the spatial organization of RBP networks to drive RNA splicing.

Position-specific binding of FUS to nascent RNA regulates mRNA length.

More than half of all human genes produce prematurely terminated polyadenylated short mRNAs. However, the underlying mechanisms remain largely elusive. CLIP-seq (cross-linking immunoprecipitation [CLIP] combined with deep sequencing) of FUS (fused in sarcoma) in neuronal cells showed that FUS is frequently clustered around an alternative polyadenylation (APA) site of nascent RNA. ChIP-seq (chromatin immunoprecipitation [ChIP] combined with deep sequencing) of RNA polymerase II (RNAP II) demonstrated that FUS stalls RNAP II and prematurely terminates transcription. When an APA site is located upstream of an FUS cluster, FUS enhances polyadenylation by recruiting CPSF160 and up-regulates the alternative short transcript. In contrast, when an APA site is located downstream from an FUS cluster, polyadenylation is not activated, and the RNAP II-suppressing effect of FUS leads to down-regulation of the alternative short transcript. CAGE-seq (cap analysis of gene expression [CAGE] combined with deep sequencing) and PolyA-seq (a strand-specific and quantitative method for high-throughput sequencing of 3' ends of polyadenylated transcripts) revealed that position-specific regulation of mRNA lengths by FUS is operational in two-thirds of transcripts in neuronal cells, with enrichment in genes involved in synaptic activities.

Phosphate enhances reactive oxygen species production and suppresses osteoblastic differentiation.

Phosphate has been shown to work as a signaling molecule in several cells including endothelial cells and chondrocytes. However, it is largely unknown how phosphate affects osteoblastic cells. In the present study, we investigated the effects of phosphate on reactive oxygen species (ROS) production and osteoblastic differentiation in murine osteoblastic MC3T3-E1 cells. Phosphate increased production of ROS in MC3T3-E1 cells and the inhibitors of sodium-phosphate cotransporter and NADPH oxidase suppressed ROS production by phosphate. Silencing Nox1 and Nox4 also inhibited the increase of ROS by phosphate. Phosphate also decreased alkaline phosphatase activity induced by bone morphogenetic protein 2 and this inhibition was abrogated by an inhibitor of NADPH oxidase. Furthermore, phosphate decreased the expression of osteoblastic marker genes in MC3T3-E1 cells. These results indicate that phosphate suppresses osteoblastic differentiation at least in part by enhancing ROS production in MC3T3-E1 cells.

[A cross-sectional study of renal function based on estimated glomerular filtration rate and urinary protein levels related to risk factors for cardiovascular events and metabolic syndrome in local residents].

OBJECTIVES: In a cross-sectional study, we investigated renal function based on estimated glomerular filtration rate (eGFR) and urinary protein levels from Specific Health Examinations in Kitakyushu city related to risk factors for cardiovascular events and metabolic syndrome in residents. METHODS: For this study, 21,625 citizens (male/female=8,637/12,988) of Kitakyushu city were investigated. Citizens were enrolled in national health insurance and data were collected from a database classified for "Specific Health Guidance" by the Kokura Medical Association health testing and services center in 2010. RESULTS: As a whole, the stage of CKD increased with age, especially among those aged 70-74 years; 32% were at CKD stage 3. Only 11% of the CKD stage 3 group had a positive urinary protein (UP) test. Subjects in stages 3-5 CKD had a higher ratio of abdominal obesity, higher systolic and diastolic blood pressure, increased fasting blood glucose, HbA1c, and fasting triglyceride levels, and lower levels of HDL-C in comparison to subjects with CKD in stages 1-2. These factors increase the complication ratio of MetS for subjects in stages 3-5. The group with a history of stroke or heart disease had a significantly lower eGFR. CONCLUSION: There is a strong relationship between CKD and risk factors for cardiovascular events and MetS. It has been indicated that lifestyle modifications, suggested by primary care doctors, are very important for the early prevention of CKD. A new preventive CKD system in Kitakyushu city, based on a Specific Health Examination, began during the fiscal year 2011, and this system is expected to decrease the incidence of end-stage renal disease and cardiovascular events.

Meclozine Attenuates the MARK Pathway in Mammalian Chondrocytes and Ameliorates FGF2-Induced Bone Hyperossification in Larval Zebrafish.

Meclozine has been developed as an inhibitor of fibroblast growth factor receptor 3 (FGFR3) to treat achondroplasia (ACH). Extracellular signal regulated kinase (ERK) phosphorylation was attenuated by meclozine in FGF2-treated chondrocyte cell line, but the site of its action has not been elucidated. Although orally administered meclozine promoted longitudinal bone growth in a mouse model of ACH, its effect on craniofacial bone development during the early stage remains unknown. Herein, RNA-sequencing analysis was performed using murine chondrocytes from FGF2-treated cultured tibiae, which was significantly elongated by meclozine treatment. Gene set enrichment analysis demonstrated that FGF2 significantly increased the enrichment score of mitogen-activated protein kinase (MAPK) family signaling cascades in chondrocytes; however, meclozine reduced this enrichment. Next, we administered meclozine to FGF2-treated larval zebrafish from 8 h post-fertilization (hpf). We observed that FGF2 significantly increased the number of ossified vertebrae in larval zebrafish at 7 days post-fertilization (dpf), while meclozine delayed vertebral ossification in FGF2-induced zebrafish. Meclozine also reversed the FGF2-induced upregulation of ossified craniofacial bone area, including ceratohyal, hyomandibular, and quadrate. The current study provided additional evidence regarding the inhibitory effect of meclozine on the FGF2-induced upregulation of MAPK signaling in chondrocytes and FGF2-induced development of craniofacial and vertebral bones.

CUGBP1 and MBNL1 preferentially bind to 3' UTRs and facilitate mRNA decay.

CUGBP1 and MBNL1 are developmentally regulated RNA-binding proteins that are causally associated with myotonic dystrophy type 1. We globally determined the in vivo RNA-binding sites of CUGBP1 and MBNL1. Interestingly, CUGBP1 and MBNL1 are both preferentially bound to 39 UTRs. Analysis of CUGBP1- and MBNL1-bound 39 UTRs demonstrated that both factors mediate accelerated mRNA decay and temporal profiles of expression arrays supported this. Role of CUGBP1 on accelerated mRNA decay has been previously reported, but the similar function of MBNL1 has not been reported to date. It is well established that CUGBP1 and MBNL1 regulate alternative splicing. Screening by exon array and validation by RT-PCR revealed position dependence of CUGBP1- and MBNL1-binding sites on the resulting alternative splicing pattern. This study suggests that regulation of CUGBP1 and MBNL1 is essential for accurate control of destabilization of a broad spectrum of mRNAs as well as of alternative splicing events.

Hypophosphatemia induced by intravenous administration of saccharated ferric oxide: another form of FGF23-related hypophosphatemia.

Fibroblast growth factor 23 (FGF23) is a humoral factor that is produced by osteocytes and regulates phosphate and vitamin D metabolism. Several hypophosphatemic diseases including X-linked, autosomal dominant and autosomal recessive hypophosphatemic rickets/osteomalacia and tumor-induced rickets/osteomalacia are caused by excess actions of FGF23. These diseases are characterized by hypophosphatemia associated with impaired proximal tubular phosphate reabsorption and inappropriately low serum 1,25-dihydroxyvitamin D [1,25(OH)(2)D] levels for hypophosphatemia. Saccharated ferric oxide is widely used in Japan for iron-deficiency anemia. While it has been shown that saccharated ferric oxide induces hypophosphatemic osteomalacia, the mechanism of this hypophosphatemia remains to be clarified. We here describe three hypophosphatemic patients caused by intravenous administration of saccharated ferric oxide. Hypophosphatemia in these patients were associated with impaired renal tubular phosphate reabsorption, rather low serum 1,25(OH)(2)D and high FGF23 levels. All these biochemical features improved by the cessation of saccharated ferric oxide. These results indicate that hypophosphatemia caused by saccharated ferric oxide is another form of FGF23-related hypophosphatemia.

Determination of phosphate in seawater by CZE with on-line transient ITP.

We developed CZE with indirect UV detection for the determination of phosphate in seawater using transient ITP as an on-line concentration procedure. The following optimum conditions were established: BGE, 5 mM 2,6-pyridinedicarboxylic acid (PDC) containing 0.01% hydroxypropylmethylcellulose (HPMC) adjusted to pH 3.5; detection wavelength, 200 nm; vacuum injection period of sample, 3 s (45 nL); terminating ion solution, 500 mM MES adjusted to pH 4.0; vacuum injection period of the terminating ion solution, 30 s (450 nL); applied voltage, 30 kV with the sample inlet side as the cathode. The LOD for phosphate was 16 microg/L (PO(3-)(4) -P) at S/N of 3. The respective values of the RSD of the peak area, peak height, and migration time for phosphate were 2.6, 2.3, and 0.34%. The proposed method was applied to the determination of phosphate in a seawater certified reference material for nutrients, MOOS-1, distributed by the National Research Council of Canada (NRC). The results were very similar to certified values. The method was also applied to the determination of phosphate in coastal seawaters. The results agreed with those obtained using a conventional spectrophotometric method.