Interrelations between the efficiency of translation start sites and other sequence features of yeast mRNAs

The translation start site (TSS) plays an important role in the control of the translational efficiency and cytoplasmic stability of eukaryotic mRNAs. The efficiency of TSS recognition is known to be influenced by sequence context, and mRNAs with weak TSSs are relatively abundant. We analyzed a sample of 4113 yeast genes in a search for features that might serve to compensate for the inefficient recognition of weak TSSs by initiating ribosomes. The first feature found to correlate with variations in TSS strength is differences in the stability of secondary structure upstream and downstream of the start AUG codon. The second feature concerns the characteristics of AUG triplets found at the beginning of the coding sequence, i.e., downstream of the predicted TSS. In particular, the proximal downstream AUG lies in frame with the CDS significantly more often if the TSS itself is located in a weak context. The accuracy of TSS annotation, the possibility of polypeptide heterogeneity due to the use of alternative downstream AUGs, and the influence of related features of mRNA sequences are discussed.Communicated by C. P. Hollenberg     

An RNA electrophoretic mobility shift and mutational analysis of rnp-4f 5′-UTR intron splicing regulatory proteins in Drosophila reveals a novel new role for a dADAR protein isoform

Alternative splicing greatly enhances the diversity of proteins encoded by eukaryotic genomes, and is also important in gene expression control. In contrast to the great depth of knowledge as to molecular mechanisms in the splicing pathway itself, relatively little is known about the regulatory events behind this process. The 5′-UTR and 3′-UTR in pre-mRNAs play a variety of roles in controlling eukaryotic gene expression, including translational modulation, and nearly 4000 of the roughly 14,000 protein coding genes in Drosophila contain introns of unknown functional significance in their 5′-UTR. Here we report the results of an RNA electrophoretic mobility shift analysis of Drosophila rnp-4f 5′-UTR intron 0 splicing regulatory proteins. The pre-mRNA potential regulatory element consists of an evolutionarily-conserved 177-nt stem-loop arising from pairing of intron 0 with part of adjacent exon 2. Incubation of in vitro transcribed probe with embryo protein extract is shown to result in two shifted RNA–protein bands, and protein extract from a dADAR null mutant fly line results in only one shifted band. A mutated stem-loop in which the conserved exon 2 primary sequence is changed but secondary structure maintained by introducing compensatory base changes results in diminished band shifts. To test the hypothesis that dADAR plays a role in intron splicing regulation in vivo, levels of unspliced rnp-4f mRNA in dADAR mutant were compared to wild-type via real-time qRT-PCR. The results show that during embryogenesis unspliced rnp-4f mRNA levels fall by up to 85% in the mutant, in support of the hypothesis. Taken together, these results demonstrate a novel role for dADAR protein in rnp-4f 5′-UTR alternative intron splicing regulation which is consistent with a previously proposed model.     

Isolation of microsatellite markers for Pelophylax nigromaculata and a tentative application in detecting interspecific introgression

Twenty-seven microsatellite markers were obtained from the black-spotted frog, Pelophylax nigromaculata, using the FIASCO (Fast Isolation by AFLP of Sequences containing repeats) protocol. Genotyping of 60 individuals showed that the number of alleles ranged from 4 to 25 with an average of 12.78 alleles per locus. The observed and expected heterozygosities ranged from 0.137 to 0.956 and 0.594 to 0.948, with averages of 0.531 and 0.809, respectively. Many of these loci were successfully cross-amplified in five other ranid species. The introgression between P. nigromaculata and Pelophylax plancyi was assessed using 5 microsatellite loci on 159 individuals collected from Liaoning, northeast China. Two distinct genetic clusters (pure species) as well as seven unassigned individuals were identified by factorial correspondence analysis (FCA), NewHybrids and Structure. The unassigned individuals, which are likely the product of interspecific hybridization, indicate the usefulness of these microsatellite loci for future studies of hybridization and introgression between these two ranid species.     

Crystal structure of YihS in complex with D-mannose: structural annotation of Escherichia coli and Salmonella enterica yihS-encoded proteins to an aldose-ketose isomerase

The three-dimensional structure of a Salmonella enterica hypothetical protein YihS is significantly similar to that of N-acyl-d-glucosamine 2-epimerase (AGE) with respect to a common scaffold, an α₆/α₆-barrel, although the function of YihS remains to be clarified. To identify the function of YihS, Escherichia coli and S. enterica YihS proteins were overexpressed in E. coli, purified, and characterized. Both proteins were found to show no AGE activity but showed cofactor-independent aldose-ketose isomerase activity involved in the interconversion of monosaccharides, mannose, fructose, and glucose, or lyxose and xylulose. In order to clarify the structure/function relationship of YihS, we determined the crystal structure of S. enterica YihS mutant (H248A) in complex with a substrate (d-mannose) at 1.6 Å resolution. This enzyme-substrate complex structure is the first demonstration in the AGE structural family, and it enables us to identify active-site residues and postulate a reaction mechanism for YihS. The substrate, β-d-mannose, fits well in the active site and is specifically recognized by the enzyme. The substrate-binding site of YihS for the mannose C1 and O5 atoms is architecturally similar to those of mutarotases, suggesting that YihS adopts the pyranose ring-opening process by His383 and acidifies the C2 position, forming an aldehyde at the C1 position. In the isomerization step, His248 functions as a base catalyst responsible for transferring the proton from the C2 to C1 positions through a cis-enediol intermediate. On the other hand, in AGE, His248 is thought to abstract and re-adduct the proton at the C2 position of the substrate. These findings provide not only molecular insights into the YihS reaction mechanism but also useful information for the molecular design of novel carbohydrate-active enzymes with the common scaffold, α₆/α₆-barrel.     

The xanthophyll cycle and antioxidative defense system are enhanced in the wheat hybrid subjected to high light stress

Although the wheat hybrids have often shown higher grain yields, the physiological basis of the higher yields remains unknown. Previous studies suggest that tolerance to photoinhibition in the hybrid may be one of the physiological bases (Yang et al., 2006, Plant Sci 171:389-97). The objective of this study was to further investigate the possible mechanism responsible for tolerance to photoinhibition in the hybrid. Photosystem II (PSII) photochemistry, the xanthophyll cycle, and antioxidative defense system were compared between the hybrid and its parents subjected to high light stress (1500 μmol m⁻² s⁻¹). The analyses of oxygen-evolving activity, chlorophyll fluorescence, and protein blotting demonstrated that the higher tolerance in the hybrid than in its parents was associated with its higher tolerance of PSII to photoinhibition. High light induced an increase in non-photochemical quenching, and this increase was greater in the hybrid than in its parents. There were no differences in the pool size of the xanthophyll cycle between the hybrid and its parents. The content of violaxanthin decreased significantly, whereas the content of zeaxanthin + antherxanthin increased considerably during high light treatments. However, the decrease in violaxanthin content and the increase in zeaxanthin + antherxanthin content were greater in the hybrid than in its parents. High light resulted in a significant accumulation of H₂O₂, O₂⁻ and catalytic Fe, and this accumulation was less in the hybrid than in its parents. High light induced a significant increase in the activities of superoxide dismutase, catalase, ascorbate peroxidase, glutathione reductase, dehydroascorbate reductase, and monodehydroascorbate reductase, and these increases were greater in the hybrid than its parents. These results suggest that the higher tolerance to photoinhibition in the hybrid may be associated with its higher capacity for antioxidative defense metabolism and the xanthophyll cycle.     

MicroRNA-195 and microRNA-378 mediate tumor growth suppression by epigenetical regulation in gastric cancer

The epigenetic regulation of microRNAs is one of several mechanisms underlying carcinogenesis. We found that microRNA-195 (miR-195) and microRNA-378 (miR-378) were significantly down-regulated in gastric cancer tissues and gastric cancer cell lines. The expression of miR-195 and miR-378 in gastric cancer cells was significantly restored by 5-aza-dC, a demethylation reagent. The low expression of miR-195 and miR-378 was closely related to the presence of promoter CpG island methylation. Treatment with miR-195/miR-378 mimics strikingly suppressed the growth of gastric cancer cells whereas promoted the growth of normal gastric epithelial cells. In contrast, administration of miR-195/miR-378 inhibitors significantly prevented the growth of normal gastric epithelial cells. Expression of cyclin-dependent kinase 6 and vascular endothelial growth factor was down-regulated by exogenous miR-195 and miR-378, respectively. In conclusion, miR-195 and miR-378 are abnormally expressed and epigenetically regulated in gastric cancer cell lines and tissues via the suppression of CDK6 and VEGF signaling, suggesting that miR-195 and miR-378 have tumor suppressor properties in gastric cancer.     

Structural flexibility and the positive charges are the key factors in bacterial cell selectivity and membrane penetration of peptoid-substituted analog of Piscidin 1

Piscidin 1 (Pis-1) is a novel cytotoxic peptide with a cationic α-helical structure isolated from the mast cells of hybrid striped bass. In our previous study, we showed that Pis-1[PG] with a substitution of Pro⁸ for Gly⁸ in Pis-1 had higher bacterial cell selectivity than Pis-1. We designed peptoid residue-substituted peptide, Pis-1[NkG], in which Gly⁸ of Pis-1 was replaced with Nlys (Lys peptoid residue). Pis-1[NkG] had higher antibacterial activity and lower cytotoxicity against mammalian cells than Pis-1 and Pis-1[PG]. We determined the tertiary structure of Pis-1[PG] and Pis-1[NkG] in the presence of DPC micelles by NMR spectroscopy. Both peptides had a three-turn helix in the C-terminal region and a bent structure in the center. Pis-1[PG] has a rigid bent structure at Pro⁸ whereas Pis-1[NkG] existed as a dynamic equilibrium of two conformers with a flexible hinge structure at Nlys⁸. Depolarization of the membrane potential of Staphylococcus aureus and confocal laser-scanning microscopy study revealed that Pis-1[NkG] effectively penetrated the bacterial cell membrane and accumulated in the cytoplasm, whereas Pis-1[PG] did not penetrate the membrane but remained outside or on the cell surface. Introduction of a lysine peptoid at position 8 of Pis-1 provided conformational flexibility and increased the positive charge at the hinge region; both factors facilitated penetration of the bacterial cell membrane and conferred bacterial cell selectivity on Pis-1[NkG].     

Significant relationships among frost tolerance and net photosynthetic rate,water use efficiency and dehydrin accumulation in cold-treated winter oilseed rapes

Five winter oilseed rape cultivars (Benefit, Californium, Cortes, Ladoga, Navajo) were subjected to 30 days of cold treatment (4 °C) to examine the effect of cold on acquired frost tolerance (FT), dehydrin (DHN) content, and photosynthesis-related parameters. The main aim of this study was to determine whether there are relationships between FT (expressed as LT₅₀ values) and the other parameters measured in the cultivars. While the cultivar Benefit accumulated two types of DHNs (D45 and D35), the other cultivars accumulated three additional DHNs (D97, D47, and D37). The similar-sized DHNs (D45 and D47) were the most abundant; the others exhibited significantly lower accumulations. The highest correlations were detected between LT₅₀ and DHN accumulation (r = −0.815), intrinsic water use efficiency (WUEi; r = −0.643), net photosynthetic rate (r = −0.628), stomatal conductance (r = 0.511), and intracellular/intercellular CO₂ concentration (r = 0.505). Those cultivars that exhibited higher Pn rate in cold (and further a significant increase in WUEi) had higher levels of DHNs and also higher FT. No significant correlation was observed between LT₅₀ and E, PRI, or NDVI. Overall, we have shown the selected physiological parameters to be able to distinguish different FT cultivars of winter oilseed rape.