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.     

Thermal response in murine L929 cells lacking αB-crystallin expression and αB-crystallin expressing L929 transfectants

We investigated the role of B-crystallin expression in the development of thermotolerance in murine L929 cells. An initial heat-shock of 10 min at 45°C induced thermotolerance in these cells to a heat challenge at 45°C administered 24 h later. The thermotolerance ratio at 10^–1 isosurvival was 1.7. Expression of B-crystallin gene was not detected during the 24 h incubation at 37°C following heat shock by either northern or western blots. In contrast, inducible HSP70 synthesis was observed during this time period. Thus, this cell line provided an unique system in which to examine the effects of transfected B-crystallin on thermoresistance and thermotolerance. Cells stably transfected with B-crystallin under the control of an inducible promoter did not show a significant increase in the ability to develop thermotolerance. However, a stably transfected L929 clone expressing high levels of constitutive B-crystallin exhibited an approximately 50% increase in thermal resistance over parental and control cells. Though expression of B-crystallin is not requisite for the development of thermotolerance in L929 cells, overexpression of transfected B-crystallin can contribute to increased thermoresistance.     

Gene silencing with RNA interference in the human pathogenic fungus Aspergillus fumigatus

Aspergillus fumigatus is an opportunistic pathogenic fungus which causes fatal invasive aspergillosis among immunocompromised patients. To obtain a better understanding of the key elements involved in A. fumigatus virulence and to identify possible drug targets, it is necessary to be able to generate gene-deletion strains. Unfortunately, the molecular techniques available do not include a rapid method to disrupt and identify essential genes. RNA interference, a process in which the presence of double-stranded RNA homologous to a gene of interest results in specific degradation of the corresponding message, has been successfully tested on A. fumigatus. We have shown that expression of double stranded RNA corresponding to portions of the ALB1/PKSP and FKS1 genes results in reduced mRNA levels for those genes, with phenotypic consequences similar to that of gene disruption. The two genes could also be subjected to simultaneous interference through expression of chimeric double-stranded RNA. Use of RNA interference in Aspergillus will allow easier examination of the phenotypic consequences of reducing expression of a gene of interest, especially for essential genes.     

Characterization of a lymphoblastoid line deleted for lambda immunoglobulin genes

While characterizing the cat eye syndrome (CES) supernumerary chromosome for the presence of immunoglobulin gene region sequences, a lymphoblastoid cell line from one CES patient was identified in which there was selection of cells deleted for some IGLC and IGLV genes. Two distinct deletions, one on each chromosome 22, were identified, presumably arising from independent somatic recombination events occuring during B-lymphocyte differentiation. The extent of the deleted region was determined using probes from the various IGLV subgroups and they each cover at least 82 kilobases. The precise definition of the deletions was not possible because of conservation of some restriction sites in the IGLV region. The cell line was used to map putative IGLV genes within the recombinant phage V135 to the distal part of the IGLV gene region. Since the deletions are relatively small, the cell line will be valuable for mapping IGLV genes in the distal part of this region.     

Purification and properties of dehydroascorbate reductase from spinach leaves

Dehydroascorbate reductase was detected in the leaves of several plants and has been partially purified from spinach leaves. The enzyme has a MW of ca 25 000, a pH optimum of 7.5, a K_m for glutathione (GSH) of 4.43 ± 0.4 mM and a K_m for dehydroascorbate of 0.34 ± 0.05 mM. High concentrations of dehydroascorbate inhibit the enzyme. Cysteine cannot replace GSH as a donor. The purified dehydroascorbate reductase is extremely unstable and also inhibited by compounds which react with thiol groups. Dehydroascorbate does not protect the enzyme against such inhibition. GSH reduces dehydroascorbate non-enzymically at alkaline pH values.     

The presence of glutathione and glutathione reductase in chloroplasts: A proposed role in ascorbic acid metabolism

Both glutathione and an NADPH-dependent glutathione reductase are present in spinach (Spinacia oleracea L.) chloroplasts. It is proposed that glutathione functions to stabilise enzymes of the Calvin cycle, and it may also act to keep ascorbic acid in chloroplasts in the reduced form.Abbreviations GSH tripeptide glutathione - GSH reduced form of glutathione - GSSG oxidised form of glutathione     

Novel morphology in Enteromorpha (Ulvophyceae) forming green tides

"Green tides" are vast accumulations of unattached green macroalgae associated with eutrophicated marine environments. They have major ecological and economic impacts globally, so an understanding of their origin and persistence is required in order to make management decisions. Blooms predominantly consist of two common fouling genera of the Ulvales, Ulva (distromatic sheets) and Enteromorpha (monostromatic tubes). In the Baltic Sea and elsewhere green tides have increased over the last few decades. On the west coast of Finland, summer blooms consist of monostromatic sheets resembling Monostroma (Codiolales). We identified these as Enteromorpha intestinalis by comparative analyses of rDNA internal transcribed spacer 1 (ITS1), 5.8S, and ITS2 sequences, the first time monostromatic sheets have been found in the genus Enteromorpha. Ordinary attached E. intestinalis sporulated freely in culture, but the sheets reproduced only by cell regeneration into typical tubular thalli. The ITS sequences were identical to those of attached E. intestinalis populations in southwestern Finland, but differed by two substitutions from other Baltic sequences. We infer that this bloom originated from local attached populations and now reproduces clonally by fragmentation. This study provides further evidence of radical changes in gross morphology of green algae under eutrophicated conditions and the need for molecular identification.