RBP Image Database: A resource for the systematic characterization of the subcellular distribution properties of human RNA binding proteins.

RNA binding proteins (RBPs) are central regulators of gene expression implicated in all facets of RNA metabolism. As such, they play key roles in cellular physiology and disease etiology. Since different steps of post-transcriptional gene expression tend to occur in specific regions of the cell, including nuclear or cytoplasmic locations, defining the subcellular distribution properties of RBPs is an important step in assessing their potential functions. Here, we present the RBP Image Database, a resource that details the subcellular localization features of 301 RBPs in the human HepG2 and HeLa cell lines, based on the results of systematic immuno-fluorescence studies conducted using a highly validated collection of RBP antibodies and a panel of 12 markers for specific organelles and subcellular structures. The unique features of the RBP Image Database include: (i) hosting of comprehensive representative images for each RBP-marker pair, with ∼250,000 microscopy images; (ii) a manually curated controlled vocabulary of annotation terms detailing the localization features of each factor; and (iii) a user-friendly interface allowing the rapid querying of the data by target or annotation. The RBP Image Database is freely available at https://rnabiology.ircm.qc.ca/RBPImage/.

Successful management of an intraoperative electric burn broken ureteral stent: A case report.

In the treatment of some gynecological diseases, ureteral stents (double J stents) have been generally utilized in the prevention of ureteral injury. Nevertheless, if the ureteral stent is retained as a protective reminder during gynecological surgery, severe ureteral injury can be avoided. Hence, it is very essential to be familiar with the anatomy of ureter in gynecological surgery to prevent complications and morbidity. We demonstrate a case of a 49-year-old woman who presented with an electric burn broken ureteral stent in the gynecological surgery, but the ureter is only burned but not broken. This resulted in no abnormality being found during surgery. So, ureteroscopy is necessary to extract the ureteral stent and the patient is inserted with another new ureteral stent to repair the ureteral injury. The electric burn broken ureteral stent is difficult for discovering during the operation. And ureteroscopy is very crucial for the special situation of the ureteral injury.

Successful management of the stone inside Allium bulbar urethral stent: A very interesting and rare case.

The stone inside the Allium bulbar urethral stent for treatment of urethral stenosis is an exceedingly rare disease. Herein, we report a case of the stone inside the Allium bulbar urethral stent(BUS) for treating urethral stricture in a 48-year-old Chinese male patient. The patient underwent a cystoscopy and URS for the stone inside BUS. The patient had only a symptom of urodynia. Urination of the patient is unobstructed after removing BUS and the urethral stricture of the patient was cured.

A large-scale binding and functional map of human RNA-binding proteins.

Many proteins regulate the expression of genes by binding to specific regions encoded in the genome1. Here we introduce a new data set of RNA elements in the human genome that are recognized by RNA-binding proteins (RBPs), generated as part of the Encyclopedia of DNA Elements (ENCODE) project phase III. This class of regulatory elements functions only when transcribed into RNA, as they serve as the binding sites for RBPs that control post-transcriptional processes such as splicing, cleavage and polyadenylation, and the editing, localization, stability and translation of mRNAs. We describe the mapping and characterization of RNA elements recognized by a large collection of human RBPs in K562 and HepG2 cells. Integrative analyses using five assays identify RBP binding sites on RNA and chromatin in vivo, the in vitro binding preferences of RBPs, the function of RBP binding sites and the subcellular localization of RBPs, producing 1,223 replicated data sets for 356 RBPs. We describe the spectrum of RBP binding throughout the transcriptome and the connections between these interactions and various aspects of RNA biology, including RNA stability, splicing regulation and RNA localization. These data expand the catalogue of functional elements encoded in the human genome by the addition of a large set of elements that function at the RNA level by interacting with RBPs.

The miR-183/182/96 cluster functions as a potential carcinogenic factor and prognostic factor in kidney renal clear cell carcinoma.

Kidney renal clear cell carcinoma (KIRC) is the most common type of renal cell carcinoma. While a number of treatments have been developed over the past few decades, the prognosis of patients with KIRC remains poor due to tumor metastasis and recurrence. Therefore, the molecular mechanisms of KIRC require to be elucidated in order to identify novel biomarkers. MicroRNAs (miRNAs/miRs) have been studied as important regulators of gene expression in a variety of cancer types. In the present study, a bioinformatics analysis of differentially expressed miRNAs in KIRC vs. normal tissues was performed based on raw miRNA expression data and patient information downloaded from the The Cancer Genome Atlas database. Furthermore, the clinical significance of differentially expressed miRNAs was evaluated, and their target genes and biological effects were further predicted. After applying the cut-off criteria of an absolute fold change of ≥2 and P<0.05, 127 differentially expressed miRNAs between KIRC and normal tissues were identified. The product of the miR-183/182/96 gene cluster, namely miR-183, miR-96 and miR-182, was revealed to be associated with multiple clinicopathological features of KIRC and to have a significant predictive and prognostic value. Subsequent functional enrichment analysis indicated that the target genes of the three miRNAs are associated with various Panther pathways, including the α-adrenergic receptor signaling pathway, metabotropic glutamate receptor group I pathway, histamine H1 receptor-mediated signaling pathway and thyrotropin-releasing hormone receptor signaling pathway. In addition, major enriched gene ontology terms in the category biological process included the intracellular signaling cascade, cellular macromolecule catabolic process and response to DNA damage stimulus. Taken together, the present study suggested that miR-183, miR-96 and miR-182 may function as potential carcinogenic factors in KIRC and may be utilized as prognostic predictors.

Correction to: DNA copy number evolution in Drosophila cell lines.

Following publication of the original article [1], the authors reported the following errors.

Regulation of RNA editing by RNA-binding proteins in human cells.

Adenosine-to-inosine (A-to-I) editing, mediated by the ADAR enzymes, diversifies the transcriptome by altering RNA sequences. Recent studies reported global changes in RNA editing in disease and development. Such widespread editing variations necessitate an improved understanding of the regulatory mechanisms of RNA editing. Here, we study the roles of >200 RNA-binding proteins (RBPs) in mediating RNA editing in two human cell lines. Using RNA-sequencing and global protein-RNA binding data, we identify a number of RBPs as key regulators of A-to-I editing. These RBPs, such as TDP-43, DROSHA, NF45/90 and Ro60, mediate editing through various mechanisms including regulation of ADAR1 expression, interaction with ADAR1, and binding to Alu elements. We highlight that editing regulation by Ro60 is consistent with the global up-regulation of RNA editing in systemic lupus erythematosus. Additionally, most key editing regulators act in a cell type-specific manner. Together, our work provides insights for the regulatory mechanisms of RNA editing.

Vex-seq: high-throughput identification of the impact of genetic variation on pre-mRNA splicing efficiency.

Understanding the functional impact of genomic variants is a major goal of modern genetics and personalized medicine. Although many synonymous and non-coding variants act through altering the efficiency of pre-mRNA splicing, it is difficult to predict how these variants impact pre-mRNA splicing. Here, we describe a massively parallel approach we use to test the impact on pre-mRNA splicing of 2059 human genetic variants spanning 110 alternative exons. This method, called variant exon sequencing (Vex-seq), yields data that reinforce known mechanisms of pre-mRNA splicing, identifies variants that impact pre-mRNA splicing, and will be useful for increasing our understanding of genome function.

-NOD Mice Having a Lyn Tyrosine Kinase Mutation Exhibit Abnormal Neutrophil Chemotaxis.

Neutrophils from NOD (Non-Obese Diabetic) mice exhibited reduced migration speed, decreased frequency of directional changes, and loss of directionality during chemotaxis (compared to wild-type [WT] C57BL/6 mice). Additionally, F-actin of chemotaxing NOD neutrophils failed to orient toward the chemoattractant gradient and NOD neutrophil adhesion was impaired. A point mutation near the autophosphorylation site of Lyn in NOD mice was identified. Point mutations of G to A (G1412 in LynA and G1199 in LynB) cause a change of amino acid E393 (glutamic acid) to K (lysine) in LynA (E393 →K) (E372 of LynB), affecting fMLP-induced tyrosine phosphorylation. These data indicate that the Lyn mutation in NOD neutrophils is likely responsible for dysregulation of neutrophil adhesion and directed migration, implying the role of Lyn in modulating diabetic patient's susceptibility to bacterial and fungal infections. J. Cell. Physiol. 232: 1689-1695, 2017. © 2016 Wiley Periodicals, Inc.

A benchmark for RNA-seq quantification pipelines.

Obtaining RNA-seq measurements involves a complex data analytical process with a large number of competing algorithms as options. There is much debate about which of these methods provides the best approach. Unfortunately, it is currently difficult to evaluate their performance due in part to a lack of sensitive assessment metrics. We present a series of statistical summaries and plots to evaluate the performance in terms of specificity and sensitivity, available as a R/Bioconductor package ( http://bioconductor.org/packages/rnaseqcomp ). Using two independent datasets, we assessed seven competing pipelines. Performance was generally poor, with two methods clearly underperforming and RSEM slightly outperforming the rest.

Resources for the Comprehensive Discovery of Functional RNA Elements.

Transcriptome-wide maps of RNA binding protein (RBP)-RNA interactions by immunoprecipitation (IP)-based methods such as RNA IP (RIP) and crosslinking and IP (CLIP) are key starting points for evaluating the molecular roles of the thousands of human RBPs. A significant bottleneck to the application of these methods in diverse cell lines, tissues, and developmental stages is the availability of validated IP-quality antibodies. Using IP followed by immunoblot assays, we have developed a validated repository of 438 commercially available antibodies that interrogate 365 unique RBPs. In parallel, 362 short-hairpin RNA (shRNA) constructs against 276 unique RBPs were also used to confirm specificity of these antibodies. These antibodies can characterize subcellular RBP localization. With the burgeoning interest in the roles of RBPs in cancer, neurobiology, and development, these resources are invaluable to the broad scientific community. Detailed information about these resources is publicly available at the ENCODE portal (https://www.encodeproject.org/).

Distal Alternative Last Exons Localize mRNAs to Neural Projections.

Spatial restriction of mRNA to distinct subcellular locations enables local regulation and synthesis of proteins. However, the organizing principles of mRNA localization remain poorly understood. Here we analyzed subcellular transcriptomes of neural projections and soma of primary mouse cortical neurons and two neuronal cell lines and found that alternative last exons (ALEs) often confer isoform-specific localization. Surprisingly, gene-distal ALE isoforms were four times more often localized to neurites than gene-proximal isoforms. Localized isoforms were induced during neuronal differentiation and enriched for motifs associated with muscleblind-like (Mbnl) family RNA-binding proteins. Depletion of Mbnl1 and/or Mbnl2 reduced localization of hundreds of transcripts, implicating Mbnls in localization of mRNAs to neurites. We provide evidence supporting a model in which the linkage between genomic position of ALEs and subcellular localization enables coordinated induction of localization-competent mRNA isoforms through a post-transcriptional regulatory program that is induced during differentiation and reversed in cellular reprogramming and cancer.

DNA copy number evolution in Drosophila cell lines.

BACKGROUND: Structural rearrangements of the genome resulting in genic imbalance due to copy number change are often deleterious at the organismal level, but are common in immortalized cell lines and tumors, where they may be an advantage to cells. In order to explore the biological consequences of copy number changes in the Drosophila genome, we resequenced the genomes of 19 tissue-culture cell lines and generated RNA-Seq profiles. RESULTS: Our work revealed dramatic duplications and deletions in all cell lines. We found three lines of evidence indicating that copy number changes were due to selection during tissue culture. First, we found that copy numbers correlated to maintain stoichiometric balance in protein complexes and biochemical pathways, consistent with the gene balance hypothesis. Second, while most copy number changes were cell line-specific, we identified some copy number changes shared by many of the independent cell lines. These included dramatic recurrence of increased copy number of the PDGF/VEGF receptor, which is also over-expressed in many cancer cells, and of bantam, an anti-apoptosis miRNA. Third, even when copy number changes seemed distinct between lines, there was strong evidence that they supported a common phenotypic outcome. For example, we found that proto-oncogenes were over-represented in one cell line (S2-DRSC), whereas tumor suppressor genes were under-represented in another (Kc167). CONCLUSION: Our study illustrates how genome structure changes may contribute to selection of cell lines in vitro. This has implications for other cell-level natural selection progressions, including tumorigenesis.

Targeted gene deletion of prolyl hydroxylase domain protein 3 triggers angiogenesis and preserves cardiac function by stabilizing hypoxia inducible factor 1 alpha following myocardial infarction.

The key oxygen sensing molecules, Prolyl-hydroxylase domain 1-3 enzymes (PHD1-3), regulate hypoxia-inducible factor (HIF) under hypoxia. In the settings of cardiomyopathy and ischemia-reperfusion injury, PHD3 expression is elevated, resulting in decreased HIF activation. The role of PHD3 in myocardial injury is poorly understood. Hence, we aimed to determine the effects of PHD3 deletion in mice on HIF-1α and other related pathways following myocardial infarction (MI). Left coronary artery (LAD) in both wild type and prolyl hydroxylase 3 knock out (PHD3⁻/⁻) mice was ligated to induce myocardial infarction. Electrophoretic mobility shift analysis showed significant increase in DNA-binding activity of HIF-1α in PHD3⁻/⁻ mice as compared to wild type (WT) mice post MI. The PHD3⁻/⁻MI group also showed decreased fibrosis. Seven days after MI, enhanced capillary/arteriolar density was observed compared to WTMI group. PHD3⁻/⁻ mice subjected to MI also showed improved cardiac functions (Ejection fraction and Fractional shortening), as assessed by echocardiogram, compared to WT. Western blot analysis showed increased VEGF, Ang-1 & Bcl-2 expression in PHD3⁻/⁻MI group. In conclusion, ablation of the PHD3 gene resulted in increased angiogenesis and cardiac function after infarction thereby offering a potential target for pharmacological management of ischemic myocardial disease.

Cardioprotective efficacy of a novel antioxidant mix VitaePro against ex vivo myocardial ischemia-reperfusion injury.

Circumstantial evidence frequently implicates oxygen-derived free radicals and oxidative stress as mediators of myocardial ischemia/reperfusion (I/R) injury. Therefore, external supplementation of natural antioxidants plays a main role as cardioprotective compounds. This study was designed to evaluate the cardioprotective effect of VitaePro (70 mg/kg body weight, 21 days), a novel antioxidant mix of astaxanthin, lutein and zeaxanthin in a rat ex vivo model of ischemia/reperfusion injury. The cardioprotective effect of VitaePro was also compared with vitamin E (70 mg/kg body weight, 21 days) treatment. Rats were randomized into control I/R (CIR), VitaePro I/R (VPIR) and Vitamin E I/R (VEIR). After 21 days of oral treatment, isolated hearts from each group were subjected to 30 min of ischemia followed by 2 h of reperfusion. In the VPIR group compared to CIR and VEIR groups at 2 h of reperfusion, increased left ventricular functional recovery, such as left ventricular developed pressure (92.7 ± 0.7 vs. 85.3 ± 0.3 and 89.4 ± 1.2 mm Hg), dp/dt max (2518.7 ± 77.9 vs. 1962.5 ± 24 and 2255.7 ± 126.6 mm Hg/s), and aortic flow (21.5 ± 1.36 vs. 4.4 ± 0.6 and 13.2 ± 1.02 ml/min) were observed. The infarct size (27.68 ± 1.7 vs. 45.4 ± 1.8 and 35.4 ± 0.6%), apoptotic cardiomyocytes (61.7 ± 10.6 vs. 194.1 ± 14.8 and 118.7 ± 15.4 counts/100 HPF) and thiobarbituric acid reactive substances levels (80 ± 3 vs. 127 ± 5 and 103 ± 2 nM/mg tissue) also were decreased in VPIR group when compared to CIR and VEIR. As evidenced by the data, administration of vitamin E offered substantial cardioprotection to I/R injury, but VitaePro enhanced cardioprotection significantly more than vitamin E treatment. Taken in concert, the results of this study suggests that the oral ingestion of VitaePro protects myocardium from ischemia/reperfusion injury by decreasing oxidative stress and apoptosis, which may be of therapeutic benefit in the treatment of cardiovascular complications. However, further in vivo animal and human intervention studies are warranted before establishing any recommendations about usage of VitaePro for human cardiovascular complications.

Safety of oligonol, a highly bioavailable lychee-derived polyphenolic antioxidant, on liver, kidney and heart function in rats.

Oligonol (OLG), derived from lychee fruit, is a novel compound produced from the oligomerization of polyphenols. In this study, the acute effect of OLG treatment was investigated on heart, liver and kidney in rats. OLG treatment at two different doses (15 or 30 mg/kg body weight) and two different time points (1 day or 7 days of treatment) demonstrated that no toxic effects were observed on heart, liver and renal functions. Moreover, OLG did not induce any DNA damage or oxidative stress as measured by 8-hydroxy-2'-deoxyguanosine levels in plasma. OLG supplementation increased the phosphorylation of myocardial endothelial nitric oxide (NO) level (p-eNOS) in both the treatment groups. Even the low dose OLG treatment (15mg/kg b.w) demonstrated an increase in p-eNOS/eNOS ratio after normalization of p-eNOS values with eNOS on day 1 (1.5-fold) and day 7 (2.2-fold) groups as compared to control. The above results suggest that OLG treatment increases endothelial NO levels and may play a role in NO-mediated vasodilatory effects without adverse side effects on cardiovascular function. This endothelial NO production may underlie the beneficial effect of OLG in cardiovascular health.