No abstract

The explosion in sequencing technologies has provided us with an instrument to describe mammalian transcriptomes at unprecedented depths. This has revealed that alternative splicing is used extensively not only to generate protein diversity, but also as a means to regulate gene expression post-transcriptionally. Intron retention (IR) is overwhelmingly perceived as an aberrant splicing event with little or no functional consequence. However, recent work has now shown that IR is used to regulate a specific differentiation event within the haematopoietic system by coupling it to nonsense-mediated mRNA decay (NMD). Here, we highlight how IR and, more broadly, alternative splicing coupled to NMD (AS-NMD) can be used to regulate gene expression and how this is deregulated in disease. We suggest that the importance of AS-NMD is not restricted to the haematopoietic system but that it plays a prominent role in other normal and aberrant biological settings.

During transcription, most eukaryotic genes generate multiple alternative cleavage and polyadenylation (APA) sites, leading to the production of transcript isoforms with variable lengths in the 3’ untranslated region (3’UTR). In contrast to somatic cells, male germ cells, especially pachytene spermatocytes and round spermatids, express a distinct reservoir of mRNAs with shorter 3’UTRs that are essential for spermatogenesis and male fertility. However, the mechanisms underlying the enrichment of shorter 3’UTR transcripts in the developing male germ cells remain unknown. Here, we report that UPF2-mediated nonsense-mediated mRNA decay (NMD) plays an essential role in male germ cells by eliminating ubiquitous genes-derived, longer 3’UTR transcripts, and that this role is independent of its canonical role in degrading “premature termination codon” (PTC)-containing transcripts in somatic cell lineages. This report provides physiological evidence supporting a noncanonical role of the NMD pathway in achieving global 3’UTR shortening in the male germ cells during spermatogenesis.

Purpose:The purpose of this study was to examine the tumor specificity, cytotoxicity, and granulocyte macrophage colony-stimulating factor expression of CG0070, a conditionally replicating oncolytic adenovirus, in human bladder transitional cell carcinoma (TCC) cell lines and determine its antitumor efficacy in bladderTCC tumor models. Experimental Design: Virus yield and cytotoxicity assays were used to determine tumor specificity and virus replication-mediated cytotoxicity of CG0070 in a panel of human bladderTCC cell lines and primary cells in vitro. Two s.c. and one orthotopic bladderTCC xenograft tumor models were used to assess antitumor activity of CG0070. Results: In a matched isogenic pair of cell lines with differing retinoblastoma (Rb) pathway status, CG0070 showed selective E1a and granulocyte macrophage colony-stimulating factor (GM-CSF) expression in Rb pathway^defective cells. CG0070 replicated in Rb-defective bladderTCC cell lines as efficiently as wild-type adenovirus but produced 100-fold less virus in normal human cells. CG0070 was up to 1,000-fold more cytotoxic in Rb pathway^defective bladder TCC cells in comparison with normal human cells. Antitumor activity of CG0070 was shown in two bladder TCC s.c. xenograft tumor models following intratumoral injections and intravesical treatment in an orthotopic xenograft tumor model when compared with PBS treatment. Conclusions: In vitro and in vivo studies showed the selective replication, cytotoxicity, GM-CSF production, and antitumor efficacy of CG0070 in several bladder TCC models, suggesting a potential utility of this oncolytic agent for the treatment of bladder cancer. Further studies are warranted to show the role of human GM-CSF in the antitumor efficacy of CG0070.

BackgroundThe esophageal intraluminal baseline impedance may be used to evaluate the status of mucosa integrity. Esophageal acid exposure decreases the baseline impedance. We aimed to compare baseline impedance in patients with various reflux events and with different acid-related parameters, and investigate the relationships between epithelial histopathologic abnormalities and baseline impedance.MethodsA total of 229 GERD patients and 34 controls underwent 24-h multichannel intraluminal impedance and pH monitoring (MII–pH monitoring), gastroendoscopy, and completed a GERD questionnaire (GerdQ). We quantified epithelial intercellular spaces (ICSs) and expression of tight junction (TJ) proteins by histologic techniques.ResultsMean baseline values in reflux esophagitis (RE) (1752 ± 1018 Ω) and non-erosive reflux disease (NERD) (2640 ± 1143 Ω) were significantly lower than in controls (3360 ± 1258 Ω; p < 0.001 and p = 0.001, respectively). Among NERD subgroups, mean baselines in the acid reflux group (2510 ± 1239 Ω) and mixed acid/weakly acidic reflux group (2393 ± 1009 Ω) were much lower than in controls (3360 ± 1258 Ω; p = 0.020 and p < 0.001, respectively). The mean baseline in severe RE patients was significantly lower than in mild RE patients (LA-C/D vs. LA-A/B: 970 ± 505 Ω vs. 1921 ± 1024 Ω, p < 0.001). There was a significant negative correlation between baseline value and acid exposure time (AET) (r = −0.41, p < 0.001), and a weak but significant correlation (r = −0.20, p = 0.007) between baseline value and weakly AET. Negative correlations were observed between ICS and the baseline impedance (r = −0.637, p < 0.001) and claudin-1 and the baseline impedance (r = −0.648, p < 0.001).ConclusionsPatients with dominant acid reflux events and with longer AET have low baseline impedance. Baseline values are correlated with esophageal mucosal histopathologic changes such as dilated ICS and TJ alteration.Electronic supplementary materialThe online version of this article (doi:10.1007/s00535-012-0689-6) contains supplementary material, which is available to authorized users.

The organization of nucleosomes influences transcriptional activity by controlling accessibility of DNA binding proteins to the genome. Genome-wide nucleosome binding profiles have identified a canonical nucleosome organization at gene promoters, where arrays of well-positioned nucleosomes emanate from nucleosome-depleted regions. The mechanisms of formation and the function of canonical promoter nucleosome organization remain unclear. Here we analyze the genome-wide location of nucleosomes during zebrafish embryogenesis and show that well-positioned nucleosome arrays appear on thousands of promoters during the activation of the zygotic genome. The formation of canonical promoter nucleosome organization is independent of DNA sequence preference, transcriptional elongation, and robust RNA polymerase II (Pol II) binding. Instead, canonical promoter nucleosome organization correlates with the presence of histone H3 lysine 4 trimethylation (H3K4me3) and affects future transcriptional activation. These findings reveal that genome activation is central to the organization of nucleosome arrays during early embryogenesis.

Nitric oxide (NO) is an important negative modulator of tubuloglomerular feedback responsiveness. We recently found that macula densa expresses a-, b-, and g-splice variants of neuronal nitric oxide synthase 1 (NOS1), and NOS1b expression in the macula densa increases on a high-salt diet. This study tested whether upregulation of NOS1b expression in the macula densa affects sodium excretion and saltsensitive hypertension by decreasing tubuloglomerular feedback responsiveness. Expression levels of NOS1b mRNA and protein were 30-and five-fold higher, respectively, than those of NOS1a in the renal cortex of C57BL/6 mice. Furthermore, macula densa NO production was similar in the isolated perfused juxtaglomerular apparatus of wild-type (WT) and nitric oxide synthase 1a-knockout (NOS1aKO) mice. Compared with control mice, mice with macula densa-specific knockout of all nitric oxide synthase 1 isoforms (MD-NOS1KO) had a significantly enhanced tubuloglomerular feedback response and after acute volume expansion, significantly reduced GFR, urine flow, and sodium excretion. Mean arterial pressure increased significantly in MD-NOS1KO mice (P,0.01) but not NOS1flox/flox mice fed a high-salt diet. After infusion of angiotensin II, mean arterial pressure increased by 61.6 mmHg in MD-NOS1KO mice versus 32.0 mmHg in WT mice (P,0.01) fed a high-salt diet. These results indicate that NOS1b is a primary NOS1 isoform expressed in the macula densa and regulates the tubuloglomerular feedback response, the natriuretic response to acute volume expansion, and the development of salt-sensitive hypertension. These findings show a novel mechanism for salt sensitivity of BP and the significance of tubuloglomerular feedback response in long-term control of sodium excretion and BP.

BackgroundPlant roots are the primary site of perception and injury for saline-alkaline stress. The current knowledge of saline-alkaline stress transcriptome is mostly focused on saline (NaCl) stress and only limited information on alkaline (NaHCO3) stress is available.ResultsUsing Affymetrix® Soybean GeneChip®, we conducted transcriptional profiling on Glycine soja roots subjected to 50 mmol/L NaHCO3 treatment. In a total of 7088 probe sets, 3307 were up-regulated and 5720 were down-regulated at various time points. The number of significantly stress regulated genes increased dramatically after 3 h stress treatment and peaked at 6 h. GO enrichment test revealed that most of the differentially expressed genes were involved in signal transduction, energy, transcription, secondary metabolism, transporter, disease and defence response. We also detected 11 microRNAs regulated by NaHCO3 stress.ConclusionsThis is the first comprehensive wild soybean root transcriptome analysis under alkaline stress. These analyses have identified an inventory of genes with altered expression regulated by alkaline stress. The data extend the current understanding of wild soybean alkali stress response by providing a set of robustly selected, differentially expressed genes for further investigation.