Structures of nonsense-mediated mRNA decay factors UPF3B and UPF3A in complex with UPF2 reveal molecular basis for competitive binding and for neurodevelopmental disorder-causing mutation

UPF3 is a key nonsense-mediated mRNA decay (NMD) factor required for mRNA surveillance and eukaryotic gene expression regulation. UPF3 exists as two paralogs (A and B) which are differentially expressed depending on cell type and developmental stage and believed to regulate NMD activity based on cellular requirements. UPF3B mutations cause intellectual disability. The underlying molecular mechanisms remain elusive, as many of the mutations lie in the poorly characterized middle-domain of UPF3B. Here, we show that UPF3A and UPF3B share structural and functional homology to paraspeckle proteins comprising an RNA-recognition motif-like domain (RRM-L), a NONA/paraspeckle-like domain (NOPS-L), and extended α-helical domain. These domains are essential for RNA/ribosome-binding, RNA-induced oligomerization and UPF2 interaction. Structures of UPF2′s third middle-domain of eukaryotic initiation factor 4G (MIF4GIII) in complex with either UPF3B or UPF3A reveal unexpectedly intimate binding interfaces. UPF3B’s disease-causing mutation Y160D in the NOPS-L domain displaces Y160 from a hydrophobic cleft in UPF2 reducing the binding affinity ∼40-fold compared to wildtype. UPF3A, which is upregulated in patients with the UPF3B-Y160D mutation, binds UPF2 with ∼10-fold higher affinity than UPF3B reliant mainly on NOPS-L residues. Our characterization of RNA- and UPF2-binding by UPF3′s middle-domain elucidates its essential role in NMD.     

Subcellular localization of EGFR in esophageal carcinoma cell lines

Background: The EGF receptor is a therapeutic target in cancer cells, whereby mutations of EGFR and/or signalling members act as predictive markers. EGFR however also exhibits dynamic changes of subcellular localization, leading to STAT5 complex formation, nuclear translocation and induction of Aurora-A expression in squamous cancer cells. We previously described high EGFR and Aurora-A expression in esophageal cancer cells. Here, we investigated subcellular localization of EGFR and STAT5 in esophageal cancer cells. Results: Quantitative immunofluorescence analyses of four esophageal cancer cell lines reflecting esophageal squamous cell carcinomas (ESCC) and esophageal adenocarcinomas (EAC) revealed that the subcellular localization of EGFR was shifted from a membranous to cytoplasmic localization upon EGF-stimulation in OE21 (ESCC) cells. Thereby, EGFR in part co-localized with E-Cadherin. In parallel, phosphorylated STAT5-Tyr694 appeared to increase in the nucleus and to decrease at the cell membrane. In three additional cell lines, EGFR was only marginally (Kyse-410/ESCC; OE19/EAC) and weakly (OE33, EAC) detectable at the cell membrane. Partial co-localization of EGFR and E-Cadherin occurred in OE33 cells. Post EGF-stimulation, EGFR was detected in the cytoplasm, resembling endosomal compartments. Furthermore, OE19 and OE33 exhibited nuclear STAT5-Tyr694 phosphorylation upon EGF-stimulation. None of the four cell lines showed nuclear EGFR expression and localization. Conclusion: In contrast to other (squamous) cancer cells, activation of EGFR in esophageal squamous cancer cells does not result in nuclear translocation of EGFR. Still, the subcellular localization of EGFR may influence STAT5-associated signaling pathways in esophageal cancer cells and hence possibly also the responses to ErbB, respective EGFR-targeted therapies.     

The Susceptibility of Pseudomonas aeruginosa Strains from Cystic Fibrosis Patients to Bacteriophages

Phage therapy may become a complement to antibiotics in the treatment of chronic Pseudomonas aeruginosa infection. To design efficient therapeutic cocktails, the genetic diversity of the species and the spectrum of susceptibility to bacteriophages must be investigated. Bacterial strains showing high levels of phage resistance need to be identified in order to decipher the underlying mechanisms. Here we have selected genetically diverse P. aeruginosa strains from cystic fibrosis patients and tested their susceptibility to a large collection of phages. Based on plaque morphology and restriction profiles, six different phages were purified from “pyophage”, a commercial cocktail directed against five different bacterial species, including P. aeruginosa. Characterization of these phages by electron microscopy and sequencing of genome fragments showed that they belong to 4 different genera. Among 47 P. aeruginosa strains, 13 were not lysed by any of the isolated phages individually or by pyophage. We isolated two new phages that could lyse some of these strains, and their genomes were sequenced. The presence/absence of a CRISPR-Cas system (Clustered Regularly Interspaced Short Palindromic Repeats and Crisper associated genes) was investigated to evaluate the role of the system in phage resistance. Altogether, the results show that some P. aeruginosa strains cannot support the growth of any of the tested phages belonging to 5 different genera, and suggest that the CRISPR-Cas system is not a major defence mechanism against these lytic phages.     

Resin gathering in neotropical resin bugs (Insecta: Hemiptera: Reduviidae): Functional and comparative morphology

Apiomerini (Reduviidae: Harpactorinae) collect plant resins with their forelegs and use these sticky substances for prey capture or maternal care. These behaviors have not been described in detail and morphological structures involved in resin gathering, transfer, and storage remain virtually undocumented. We here describe these behaviors in Apiomerus flaviventris and document the involved structures. To place them in a comparative context, we describe and document leg and abdominal structures in 14 additional species of Apiomerini that represent all but one of the 12 recent genera in the tribe. Based on these morphological data in combination with the behavioral observations on A. flaviventris, we infer behavioral and functional hypotheses for the remaining genera within the tribe Apiomerini. Setal abdominal patches for resin storage are associated with maternal care so far only documented for species of Apiomerus. Based on the occurrence of these patches in several other genera, we propose that maternal care is widespread within the tribe. Ventral abdominal glands are widespread within female Apiomerini. We propose that their products may prevent hardening of stored resins thus providing long‐term supply for egg coating. Judging from the diverse setal types and arrangements on the front legs, we predict six different behavioral patterns of resin gathering within the tribe. J. Morphol., 2011. © 2010 Wiley‐Liss, Inc.     

<Emphasis Type="Italic">Sphedamnocarpus andersonii</Emphasis> (Malpighiaceae), a new species from Madagascar

Sphedamnocarpus andersonii, a new species from the Ihorombe Region of Madagascar, is described and illustrated. The twiggy, white-flowered shrubs are characterized by linear leaves, unique in the genus.     

The Development of Diet-Induced Obesity and Glucose Intolerance in C57Bl/6 Mice on a High-Fat Diet Consists of Distinct Phases

High–fat (HF) diet-induced obesity and insulin insensitivity are associated with inflammation, particularly in white adipose tissue (WAT). However, insulin insensitivity is apparent within days of HF feeding when gains in adiposity and changes in markers of inflammation are relatively minor. To investigate further the effects of HF diet, C57Bl/6J mice were fed either a low (LF) or HF diet for 3 days to 16 weeks, or fed the HF-diet matched to the caloric intake of the LF diet (PF) for 3 days or 1 week, with the time course of glucose tolerance and inflammatory gene expression measured in liver, muscle and WAT. HF fed mice gained adiposity and liver lipid steadily over 16 weeks, but developed glucose intolerance, assessed by intraperitoneal glucose tolerance tests (IPGTT), in two phases. The first phase, after 3 days, resulted in a 50% increase in area under the curve (AUC) for HF and PF mice, which improved to 30% after 1 week and remained stable until 12 weeks. Between 12 and 16 weeks the difference in AUC increased to 60%, when gene markers of inflammation appeared in WAT and muscle but not in liver. Plasma proteomics were used to reveal an acute phase response at day 3. Data from PF mice reveals that glucose intolerance and the acute phase response are the result of the HF composition of the diet and increased caloric intake respectively. Thus, the initial increase in glucose intolerance due to a HF diet occurs concurrently with an acute phase response but these effects are caused by different properties of the diet. The second increase in glucose intolerance occurs between 12 - 16 weeks of HF diet and is correlated with WAT and muscle inflammation. Between these times glucose tolerance remains stable and markers of inflammation are undetectable.     

Prevalence of the 281 (Gly→Glu) mutation in hepatoerythropoietic porphyria and porphyria cutanea tarda

Summary The prevalence of the 281 (GlyGlu) mutation in hepatoerythropoietic porphyria (HEP) was investigated by the use of hybridization with a synthetic oligonucleotide probe. The mutation was found in HEP-affected members of two unrelated families from Spain, but was absent in two other patients from Italy and Portugal who also had HEP. Moreover, this mutation was not detected in 13 unrelated cases of familial (type II) porphyria cutanea tarda.