Nuclear export of circular RNA.

Circular RNAs (circRNAs), which are increasingly being implicated in a variety of functions in normal and cancerous cells1-5, are formed by back-splicing of precursor mRNAs in the nucleus6-10. circRNAs are predominantly localized in the cytoplasm, indicating that they must be exported from the nucleus. Here we identify a pathway that is specific for the nuclear export of circular RNA. This pathway requires Ran-GTP, exportin-2 and IGF2BP1. Enhancing the nuclear Ran-GTP gradient by depletion or chemical inhibition of the major protein exporter CRM1 selectively increases the nuclear export of circRNAs, while reducing the nuclear Ran-GTP gradient selectively blocks circRNA export. Depletion or knockout of exportin-2 specifically inhibits nuclear export of circRNA. Analysis of nuclear circRNA-binding proteins reveals that interaction between IGF2BP1 and circRNA is enhanced by Ran-GTP. The formation of circRNA export complexes in the nucleus is promoted by Ran-GTP through its interactions with exportin-2, circRNA and IGF2BP1. Our findings demonstrate that adaptors such as IGF2BP1 that bind directly to circular RNAs recruit Ran-GTP and exportin-2 to export circRNAs in a mechanism that is analogous to protein export, rather than mRNA export.

Biostimulatory Micro-Fragmented Nanofiber-Hydrogel Composite Improves Mesenchymal Stem Cell Delivery and Soft Tissue Remodeling.

Functional microgels are preferred stem cell carriers due to the ease of delivery through minimally invasive injection and seamless integration with the surrounding host tissue. A biostimulatory nanofiber-hydrogel composite (NHC) has been previously developed through covalently crosslinking a hyaluronic acid hydrogel network with surface-functionalized poly (ε-caprolactone) nanofiber fragments. The NHC mimics the microarchitecture of native soft tissue matrix, showing enhanced cell infiltration, immunomodulation, and proangiogenic properties. Here, injectability of the pre-formed NHC is improved by mechanical fragmentation, making it into micro-fragmented NHC (mfNHC) in a granular gel form as a stem cell carrier to deliver mesenchymal stem cells (MSCs) for soft tissue remodeling. The mfNHC shows a similar storage modulus but a significantly reduced injection force, as compared with the corresponding bulk NHC. When injected subcutaneously in a rat model, mfNHC-MSC constructs initiate an elevated level of host macrophage infiltration, more pro-regenerative polarization, and subsequently, improved angiogenesis and adipogenesis response when compared to mfNHC alone. A similar trend of host cell infiltration and pro-angiogenic response is detected in a swine model with a larger volume injection. These results suggest a strong potential for use of the mfNHC as an injectable carrier for cell delivery and soft tissue remodeling.

HDLBP binds ER-targeted mRNAs by multivalent interactions to promote protein synthesis of transmembrane and secreted proteins.

The biological role of RNA-binding proteins in the secretory pathway is not well established. Here, we describe that human HDLBP/Vigilin directly interacts with more than 80% of ER-localized mRNAs. PAR-CLIP analysis reveals that these transcripts represent high affinity HDLBP substrates and are specifically bound in their coding sequences (CDS), in contrast to CDS/3'UTR-bound cytosolic mRNAs. HDLBP crosslinks strongly to long CU-rich motifs, which frequently reside in CDS of ER-localized mRNAs and result in high affinity multivalent interactions. In addition to HDLBP-ncRNA interactome, quantification of HDLBP-proximal proteome confirms association with components of the translational apparatus and the signal recognition particle. Absence of HDLBP results in decreased translation efficiency of HDLBP target mRNAs, impaired protein synthesis and secretion in model cell lines, as well as decreased tumor growth in a lung cancer mouse model. These results highlight a general function for HDLBP in the translation of ER-localized mRNAs and its relevance for tumor progression.

Single-molecule mRNA and translation imaging in neurons.

Memory-relevant neuronal plasticity is believed to require local translation of new proteins at synapses. Understanding this process has necessitated the development of tools to visualize mRNA within relevant neuronal compartments. In this review, we summarize the technical developments that now enable mRNA transcripts and their translation to be visualized at single-molecule resolution in both fixed and live cells. These tools include single-molecule fluorescence in situ hybridization (smFISH) to visualize mRNA in fixed cells, MS2/PP7 labelling for live mRNA imaging and SunTag labelling to observe the emergence of nascent polypeptides from a single translating mRNA. The application of these tools in cultured neurons and more recently in whole brains promises to revolutionize our understanding of local translation in the neuronal plasticity that underlies behavioural change.

Using bioinformatics approaches to investigate driver genes and identify BCL7A as a prognostic gene in colorectal cancer.

Colorectal cancer (CRC) results from the uncontrolled growth of cells in the colon, rectum, or appendix. The 5-year relative survival rate for patients with CRC is 65% and is correlated with the stage at diagnosis (being 91% for stage I at diagnosis versus 12% for stage IV). This study aimed to identify CRC driver genes to assist in the design of a cancer panel to detect gene mutations during clinical early-stage screening and identify genes for use in prognostic assessments and the evaluation of appropriate treatment options. First, we utilized bioinformatics approaches to analyze 354 paired sequencing profiles from The Cancer Genome Atlas (TCGA) to identify CRC driver genes and analyzed the sequencing profiles of 38 patients with >5 years of follow-up data to search for prognostic genes. The results revealed eight driver genes and ten prognostic genes. Next, the presence of the identified gene mutations was verified using tissue and blood samples from Taiwanese CRC patients. The results showed that the set identified gene mutations provide high coverage for driver gene screening, and APC, TP53, PIK3CA, and FAT4 could be detected in blood as ctDNA test targets. We further found that BCL7A gene mutation was correlated with prognosis in CRC (log-rank p-value = 0.02), and that mutations of BCL7A could be identified in ctDNA samples. These findings may be of value in clinical early cancer detection, disease monitoring, drug development, and treatment efforts in the future.

A Single-Molecule RNA Mobility Assay to Identify Proteins that Link RNAs to Molecular Motors.

mRNA transport and localization is a key aspect of posttranscriptional gene regulation. While the transport of many mRNAs is thought to occur through the recruitment of molecular motors, it has been a challenge to identify RNA-binding proteins (RBPs) that directly interact with motors by conventional assays. In order to identify RBPs and their specific domains that are responsible for recruiting a motor to transport granules, we have developed a single-molecule RNA mobility assay that enables quantifying the effect of a tethered RBP on the movement of an RNA. We demonstrate that tethering of RNAs to myosin or kinesin through their well-characterized interacting proteins results in quantitative differences in RNA mobility. This methodology provides a framework for identifying RBPs that mediate associations with motors.

Author Correction: CPSF3-dependent pre-mRNA processing as a druggable node in AML and Ewing's sarcoma.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

CPSF3-dependent pre-mRNA processing as a druggable node in AML and Ewing's sarcoma.

The post-genomic era has seen many advances in our understanding of cancer pathways, yet resistance and tumor heterogeneity necessitate multiple approaches to target even monogenic tumors. Here, we combine phenotypic screening with chemical genetics to identify pre-messenger RNA endonuclease cleavage and polyadenylation specificity factor 3 (CPSF3) as the target of JTE-607, a small molecule with previously unknown target. We show that CPSF3 represents a synthetic lethal node in a subset of acute myeloid leukemia (AML) and Ewing's sarcoma cancer cell lines. Inhibition of CPSF3 by JTE-607 alters expression of known downstream effectors in AML and Ewing's sarcoma lines, upregulates apoptosis and causes tumor-selective stasis in mouse xenografts. Mechanistically, it prevents the release of newly synthesized pre-mRNAs, resulting in read-through transcription and the formation of DNA-RNA hybrid R-loop structures. This study implicates pre-mRNA processing, and specifically CPSF3, as a druggable target providing an avenue to therapeutic intervention in cancer.

The structural basis for RNA selectivity by the IMP family of RNA-binding proteins.

The IGF2 mRNA-binding proteins (ZBP1/IMP1, IMP2, IMP3) are highly conserved post-transcriptional regulators of RNA stability, localization and translation. They play important roles in cell migration, neural development, metabolism and cancer cell survival. The knockout phenotypes of individual IMP proteins suggest that each family member regulates a unique pool of RNAs, yet evidence and an underlying mechanism for this is lacking. Here, we combine systematic evolution of ligands by exponential enrichment (SELEX) and NMR spectroscopy to demonstrate that the major RNA-binding domains of the two most distantly related IMPs (ZBP1 and IMP2) bind to different consensus sequences and regulate targets consistent with their knockout phenotypes and roles in disease. We find that the targeting specificity of each IMP is determined by few amino acids in their variable loops. As variable loops often differ amongst KH domain paralogs, we hypothesize that this is a general mechanism for evolving specificity and regulation of the transcriptome.

YM500v2: a small RNA sequencing (smRNA-seq) database for human cancer miRNome research.

We previously presented YM500, which is an integrated database for miRNA quantification, isomiR identification, arm switching discovery and novel miRNA prediction from 468 human smRNA-seq datasets. Here in this updated YM500v2 database (http://ngs.ym.edu.tw/ym500/), we focus on the cancer miRNome to make the database more disease-orientated. New miRNA-related algorithms developed after YM500 were included in YM500v2, and, more significantly, more than 8000 cancer-related smRNA-seq datasets (including those of primary tumors, paired normal tissues, PBMC, recurrent tumors, and metastatic tumors) were incorporated into YM500v2. Novel miRNAs (miRNAs not included in the miRBase R21) were not only predicted by three independent algorithms but also cleaned by a new in silico filtration strategy and validated by wetlab data such as Cross-Linked ImmunoPrecipitation sequencing (CLIP-seq) to reduce the false-positive rate. A new function 'Meta-analysis' is additionally provided for allowing users to identify real-time differentially expressed miRNAs and arm-switching events according to customer-defined sample groups and dozens of clinical criteria tidying up by proficient clinicians. Cancer miRNAs identified hold the potential for both basic research and biotech applications.

Preoperative chemoradiotherapy with oxaliplatin and tegafur-uracil in locally advanced rectal cancer: pathologic complete response rate and preliminary results of overall and disease-free survival in a single institute in Taiwan.

BACKGROUND: We conducted a Phase II study of biweekly oxaliplatin plus oral tegafur-uracil in the preoperative chemoradiotherapy (CRT) for locally advanced resectable mid-to-lower rectal cancer in our hospital, to evaluate the feasibility of this drug combination in tumor pathologic response, acute toxicity, local control, disease-free survival (DFS), overall survival (OS), and time to distant metastasis in an Asian cohort. METHODS: Twenty patients with histopathologically confirmed rectal cancer (Stage II-III) were enrolled in the study. Radiotherapy of 50 Gy was delivered in 25 fractions of 2 Gy, one fraction/day, five fractions/week, for 5 weeks. Oxaliplatin 55 mg/m(2) was administered intravenously for 60 minutes on Day 1 every 2 weeks, and tegafur-uracil 350 mg/m(2) was given orally everyday during the whole radiotherapy course, including holidays. Surgery was scheduled 6 weeks after completion of the preoperative chemoradiotherapy. The primary endpoint was to determine the pathologic complete response (pCR) rate after this neoadjuvant chemoradiotherapy. The secondary endpoint was to determine the treatment-related toxicity profile, local control, DFS, OS, and time to metastasis. RESULTS: All patients underwent a complete course of preoperative chemoradiotherapy. There was no local recurrence during the study period. The complete resection rate was 20/20 (100%) and the close resection margin rate was 3/20 (15%). The pCR rate was 8/20 (40%). During chemoradiotherapy, the most frequent toxicity was diarrhea 9/20 (45% of patients, grade 2 in 3/20, 15%). There were no grade 3 or higher hematologic or non-hematologic events or treatment-related deaths. The 3-year OS and DFS rates were 94.1% and 78.6%, respectively. CONCLUSION: Preoperative chemoradiotherapy with oxaliplatin and tegafur-uracil was well-tolerated and achieved an excellent pCR in our patients with locally advanced mid-to-lower rectal cancer.

Single-molecule analysis of gene expression using two-color RNA labeling in live yeast.

Live-cell imaging of mRNA yields important insights into gene expression, but it has generally been limited to the labeling of one RNA species and has never been used to count single mRNAs over time in yeast. We demonstrate a two-color imaging system with single-molecule resolution using MS2 and PP7 RNA labeling. We use this methodology to measure intrinsic noise in mRNA levels and RNA polymerase II kinetics at a single gene.

The volume of retropharyngeal nodes predicts distant metastasis in patients with advanced nasopharyngeal carcinoma.

We investigated the effect of retropharyngeal nodal volumes (RNV) on distant metastasis in patients with advanced nasopharyngeal carcinoma (NPC). From February 2000 to June 2006, a total of 181 patients with biopsy-proven NPC, no distant metastasis, and available pre-treatment magnetic resonance imaging (MRI) were retrospectively reviewed. Most of the patients (95.6%) had stage III/IV diseases. The contour of retropharyngeal nodes ≥5mm was delineated on the axial slides of pre-treatment T2-weighted MRI without contrast enhancement. The RNV was calculated by the Eclipse™ treatment planning software. The primary end-points were subsequent distant failure rates and distant metastasis failure-free survival (DMFFS). The pre-treatment RNV in patients who developed distant failure was higher than in those without distant failure (P=0.0536). The distant failure rates between the patients with RNV > and ≤4.68cm(3) were 33.3% and 16.0%, respectively (P=0.0112). The rates of 7-year DMFFS in patients with RNV > and ≤4.68cm(3) were 66.4% and 83.5%, respectively (P=0.0043). Multivariate Cox analysis showed N-stage (P<0.001), gender (P=0.026), and RNV (P=0.088) were important predictors for DMFFS. We conclude that the RNV measured by MRI is a potential predictor of distant metastasis in patients with advanced NPC.

Real-time observation of transcription initiation and elongation on an endogenous yeast gene.

Cellular messenger RNA levels are achieved by the combinatorial complexity of factors controlling transcription, yet the small number of molecules involved in these pathways fluctuates stochastically. It has not yet been experimentally possible to observe the activity of single polymerases on an endogenous gene to elucidate how these events occur in vivo. Here, we describe a method of fluctuation analysis of fluorescently labeled RNA to measure dynamics of nascent RNA--including initiation, elongation, and termination--at an active yeast locus. We find no transcriptional memory between initiation events, and elongation speed can vary by threefold throughout the cell cycle. By measuring the abundance and intranuclear mobility of an upstream transcription factor, we observe that the gene firing rate is directly determined by trans-activating factor search times.

Plasma EBV DNA clearance rate as a novel prognostic marker for metastatic/recurrent nasopharyngeal carcinoma.

PURPOSE: To investigate the prognostic effect of the concentrations and clearance rates of plasma EBV DNA in metastatic/recurrent nasopharyngeal carcinoma (NPC). EXPERIMENTAL DESIGN: Thirty relapsed and four previously nontreated metastatic NPC patients were treated according to the consensus guidelines of the head and neck cancer team in our hospital (i.v. chemotherapy first, followed by local irradiation boost and oral maintenance chemotherapy where applicable). Multiple plasma samples were collected during the first month of chemotherapy. Circulating EBV DNA concentrations were measured by a real-time quantitative PCR. The half-life values (t(1/2)) of plasma EBV DNA clearance were calculated. The associations between clinical outcome and plasma EBV DNA assays were analyzed. RESULTS: Tumor response evaluated after 12 weeks of treatment showed 14 complete responses (41.2%), 12 partial responses (35.3%), 7 stable diseases (20.6%), and 1 progression disease (2.9%). The plasma EBV DNA concentrations have no significant effects on outcome prediction. The t(1/2) of plasma EBV DNA clearance ranged from 1.85 to 28.29 days (median, 3.99). Patients with a short t(1/2) of plasma EBV DNA clearance have significantly higher complete response rate and overall survival than those with long t(1/2). Multivariate analysis revealed a significant effect of the t(1/2) of plasma EBV DNA clearance on survival. CONCLUSIONS: The clearance rates of plasma EBV DNA during the first month of chemotherapy can predict tumor response and patient survival. Early change of chemotherapy regimen may be considered for patients with slow plasma EBV DNA clearance rate.

Enteritis cystica profunda: is trauma the etiology? Interval development in the previously normal ileum: a case report and literature review.

Although well described in the colon, enteritis cystica profunda is an extremely rare lesion of the small intestine with only a few cases documented in English medical literature. It is a benign condition most often associated with Crohn's disease or Peutz-Jeghers syndrome in the adult. It has been described presenting as ileoileal intussusception in the pediatric population. We report a case of enteritis cystica profunda found incidentally on exploratory laparotomy with evidence of interval development over a 5-month period confirming trauma as the etiology.

Long-term prognostic effects of plasma epstein-barr virus DNA by minor groove binder-probe real-time quantitative PCR on nasopharyngeal carcinoma patients receiving concurrent chemoradiotherapy.

PURPOSE: To evaluate the long-term prognostic impact of plasma Epstein-Barr virus (EBV) DNA concentration measured by real-time quantitative polymerase chain reaction (RTQ-PCR) in nasopharyngeal carcinoma (NPC) patients receiving concurrent chemoradiotherapy (CCRT). METHODS AND MATERIALS: Epstein-Barr virus DNA was retrospectively measured from stock plasma of 152 biopsy-proven NPC patients with Stage II-IV (M0) disease with a RTQ-PCR using the minor groove binder-probe. All patients received CCRT with a median follow-up of 78 months. We divided patients into three subgroups: (1) low pretreatment EBV DNA (<1,500 copies/mL) and undetectable posttreatment EBV DNA (pre-L/post-U), (2) high pretreatment EBV DNA (> or =1,500 copies/mL) and undetectable posttreatment EBV DNA (pre-H/post-U), and (3) low or high pretreatment EBV DNA and detectable posttreatment EBV DNA (pre-L or H/post-D) for prognostic analyses. RESULTS: Epstein-Barr virus DNA (median concentration, 573 copies/mL; interquartile range, 197-3,074) was detected in the pretreatment plasma of 94.1% (143/152) of patients. After treatment, plasma EBV DNA decreased or remained 0 for all patients and was detectable in 31 patients (20.4%) with a median concentration 0 copy/mL (interquartile range, 0-0). The 5-year overall survival rates of the pre-L/post-U, pre-H/post-U, and pre-L or H/post-D subgroups were 87.2%, 71.0%, and 38.7%, respectively (p < 0.0001). The relapse-free survival showed similar results with corresponding rates of 85.6%, 75.9%, and 26.9%, respectively (p < 0.0001). Multivariate Cox analysis confirmed the superior effects of plasma EBV DNA compared to other clinical parameters in prognosis prediction. CONCLUSION: Plasma EBV DNA is the most valuable prognostic factor for NPC. More chemotherapy should be considered for patients with persistently detectable EBV DNA after CCRT.

Comparison of the prognostic impact of serum anti-EBV antibody and plasma EBV DNA assays in nasopharyngeal carcinoma.

PURPOSE: Nasopharyngeal carcinoma (NPC) has been proven as an Epstein-Barr virus (EBV)-associated cancer. Serum anti-EBV antibodies and plasma EBV DNA have been investigated as surrogate markers for NPC. A comparison of the prognostic impacts of both assays has never been reported. METHODS AND MATERIALS: Paired serum and plasma samples from 114 previously untreated NPC patients were collected and subjected to an immunofluorescence assay for immunoglobulin (Ig)A and IgG antibodies against the viral capsid antigen (VCA) and a real-time quantitative polymerase chain reaction assay for EBV DNA measurement. The effects of both assays on patient prognosis were thoroughly investigated. RESULTS: Relapsed patients had significantly higher pretreatment EBV DNA concentration than patients without relapse (p = 0.0006). No associations of VCA-IgA (p = 0.9669) or VCA-IgG (p = 0.6125) were observed between patients with and without relapse. The 4-year overall survival (60.3% vs. 93.1%, p < 0.0001) and relapse-free survival rates (54.4% vs. 77.9%, p = 0.0009) were significantly lower in patients with higher pretreatment EBV DNA load than in those with lower EBV DNA load. Patients with persistently detectable EBV DNA after treatment had significantly worse 4-year overall (30.8% vs. 84.6%, p < 0.0001) and relapse-free survival rates (15.4% vs. 74.0%, p < 0.0001) than those with undetectable EBV DNA. The VCA-IgA and VCA-IgG titer could not predict survivals (all p > 0.1). Cox multivariate analyses also showed the same results. CONCLUSION: Plasma EBV DNA is superior to serum EBV VCA antibodies in prognostic predictions for NPC.