Conserved elements in the 3′ untranslated regions of c-fos and actin mRNAs

In this study, the nucleotide sequences of the 3 untranslated regions (UTR) of the mouse and human c-fos genes, and the rat and human -actin genes were examined. It is shown (i) that the 3 UTR of c-fos is highly conserved between mouse and man, (ii) that multiple copies of a 12 bp element occur, in clusters, in the 3 UTR both of c-fos and of -actin. This conserved 12 bp element is analogous to the putative repressor binding site previously identified (Renan,Bioscience Reports,5 (1985), 739–753). These findings provide additional support for the proposal that regulatory signals are located in the 3 UTR's of certain genes.     

PolyC-Binding Protein 1 Interacts with 5′-Untranslated Region of Enterovirus 71 RNA in Membrane-Associated Complex to Facilitate Viral Replication

Enterovirus 71 (EV71) is one causative agent of hand, foot, and mouth disease (HFMD), which may lead to severe neurological disorders and mortality in children. EV71 genome is a positive single-stranded RNA containing a single open reading frame (ORF) flanked by 5′-untranslated region (5′UTR) and 3′UTR. The 5′UTR is fundamentally important for virus replication by interacting with cellular proteins. Here, we revealed that poly(C)-binding protein 1 (PCBP1) specifically binds to the 5′UTR of EV71. Detailed studies indicated that the RNA-binding K-homologous 1 (KH1) domain of PCBP1 is responsible for its binding to the stem-loop I and IV of EV71 5′UTR. Interestingly, we revealed that PCBP1 is distributed in the nucleus and cytoplasm of uninfected cells, but mainly localized in the cytoplasm of EV71-infected cells due to interaction and co-localization with the viral RNA. Furthermore, sub-cellular distribution analysis showed that PCBP1 is located in ER-derived membrane, in where virus replication occurred in the cytoplasm of EV71-infected cells, suggesting PCBP1 is recruited in a membrane-associated replication complex. In addition, we found that the binding of PCBP1 to 5′UTR resulted in enhancing EV71 viral protein expression and virus production so as to facilitate viral replication. Thus, we revealed a novel mechanism in which PCBP1 as a positive regulator involved in regulation of EV71 replication in the host specialized membrane-associated replication complex, which provides an insight into cellular factors involved in EV71 replication.     

PredSL： A Tool for the N-terminal Sequence-based Prediction of Protein Subcellular Localization

The ability to predict the subcellular localization of a protein from its sequence is of great importance, as it provides information about the protein＇s function. We present a computational tool, PredSL, which utilizes neural networks, Markov chains, profile hidden Markov models, and scoring matrices for the prediction of the subcellular localization of proteins in eukaryotic cells from the N-terminal amino acid sequence. It aims to classify proteins into five groups： chloroplast, thylakoid, mitochondrion, secretory pathway, and ＂other＂. When tested in a fivefold cross-validation procedure, PredSL demonstrates 86.7% and 87.1% overall accuracy for the plant and non-plant datasets, respectively. Compared with TargetP, which is the most widely used method to date, and LumenP, the results of PredSL are comparable in most cases. When tested on the experimentally verified proteins of the Saccharomyces cerevisiae genome, PredSL performs comparably if not better than any available algorithm for the same task. Furthermore, PredSL is the only method capable for the prediction of these subcellular localizations that is available as a stand-alone application through the URL： http：//bioinformatics.biol.uoa.gr/PredSL/.     

Prediction of Subcellular Localization of Apoptosis Protein Using Chou’s Pseudo Amino Acid Composition

Apoptosis proteins play an essential role in regulating a balance between cell proliferation and death. The successful prediction of subcellular localization of apoptosis proteins directly from primary sequence is much benefited to understand programmed cell death and drug discovery. In this paper, by use of Chou’s pseudo amino acid composition (PseAAC), a total of 317 apoptosis proteins are predicted by support vector machine (SVM). The jackknife cross-validation is applied to test predictive capability of proposed method. The predictive results show that overall prediction accuracy is 91.1% which is higher than previous methods. Furthermore, another dataset containing 98 apoptosis proteins is examined by proposed method. The overall predicted successful rate is 92.9%.     

Conservation of the 3′-untranslated regions of calmodulin mRNAs in cetaceans

Three separate calmodulin (CaM) genes (I, II and III) encoding an identical CaM protein but differing in the 5- and 3-untranslated regions of each of the three mRNAs are present and highly conserved in all mammals (so far examined). Primers complementary to the 3- untranslated region (3UTR) of each of the three mRNAs occurring in human, rat and mouse were synthesized and used to amplify regions of the 3UTR from genomic DNA isolated from cetaceans, specifically from the bottled-nosed dolphin (Tursiops truncates), the pygmy sperm whale (Kogia breviceps) and the humpback whale (Megaptera novaeangliae). Using several primers and PCR conditions, the three CaM genes were identified in all three species by this method with one exception. The sequenced regions of the 3UTRs of the three genes of the cetaceans exhibited a high percentage identity when compared to the corresponding regions of these three CaM mRNAs isolated from humans (85-96%). These partial sequences of the 3UTR regions and the corresponding regions for humans, rats and mice that were available from the database were aligned and a phylogenetic tree was constructed. The three CaM genes from all species showed a close phylogenetic relationship based on these 3UTR sequences. Such high conservation of the 3UTRs suggests a specialized and significant function for this region in mammals.     

Prediction of Protein–Protein Interaction Sites in Sequences and 3D Structures by Random Forests

Identifying interaction sites in proteins provides important clues to the function of a protein and is becoming increasingly relevant in topics such as systems biology and drug discovery. Although there are numerous papers on the prediction of interaction sites using information derived from structure, there are only a few case reports on the prediction of interaction residues based solely on protein sequence. Here, a sliding window approach is combined with the Random Forests method to predict protein interaction sites using (i) a combination of sequence- and structure-derived parameters and (ii) sequence information alone. For sequence-based prediction we achieved a precision of 84% with a 26% recall and an F-measure of 40%. When combined with structural information, the prediction performance increases to a precision of 76% and a recall of 38% with an F-measure of 51%. We also present an attempt to rationalize the sliding window size and demonstrate that a nine-residue window is the most suitable for predictor construction. Finally, we demonstrate the applicability of our prediction methods by modeling the Ras–Raf complex using predicted interaction sites as target binding interfaces. Our results suggest that it is possible to predict protein interaction sites with quite a high accuracy using only sequence information.     

Conserved Putative Signals in 3′ Intron Junctions in Rodents

Abstract

Several 3′ splice signals are known todate. At the 3′ splice site an AG doublet is frequently found. Just upstream of the splice site there is a string of 6–11 pyrimidines. More recently it has been found that one of the stages in the splicing process involves formation of a lariat, in which the 5′ end of the intron forms a 2′-5′ branch with an A residue located 18–37 nucleotides upstream of the 3′ splice site. The branching-point consensus is weakly defined and consists of the sequence YNYTRAY, where Y is a pyrimidine, R a purine and N any base. The A in the sixth position is the one with which branching occurs. Here we present the results of extensive searches for additional putative signals around the branching-point consensus and the 3′ splice site in rodent nuclear precursor mRNAs. The signals obtained for the over 370 rodent introns are compared with those found in a larger eukaryotic sample containing over 900 nuclear pre-mRNA introns. Of particular interest are GGGA and CCCA In both analyses GGGA occurs about 60 nucleotides upstream and CCCA is found 3–40 nucleotides downstream from the 3′ splice site. A model explaining some of the putative signals discussed here is also proposed. This model involves formation of alternate stem-loop structures around the branching point and 3′ splice site. Such signals and structures can possibly aid in protein or nucleoprotein branching point and splice site recognition.     

The Presence of 3′–5′ Exonuclease Activity in Rat Brain Neurons and Its Role in Template-Driven Extension of 3′-Mismatched Primers by DNA-Polymerase β in Aging Neurons

A three-step reaction strategy has been developed to examine the mechanism of extension of a mismatched primer in an oligoduplex substrate by rat neuronal extracts and DNA polymerase beta. The results revealed that in the case of duplexes with a mismatch at 3'-end of primer, significant extension by DNA polymerase beta has taken place only after the removal of the mismatched base, thus indicating the presence of a proof reading 3'-5' exonuclease activity in neuronal extracts of all ages. A closer examination of the neuronal exonuclease activity revealed that bases are excised from the 3' end in a sequential and nonspecific manner, although initial excision of a mismatched base was slightly faster. Further, the excision efficiency is seen to decrease with the age of the animal but apparently does not go below a critical level so as to become a rate-limiting factor for the DNA-repair activity.     

Subcellular Localization and Properties of L-Galactono-γ-lactone Dehydrogenase in Spinach Leaves

L-Galactono-γ-lactone dehydrogenase, which catalyzes the final step of the biosynthesis of L-ascorbate, is bound to spinach mitochondrial membrane, as confirmed by linear sucrose density gradient centrifugation. The solubilized enzyme was very labile, but stabilized in the presence of L-galactono-γ-lactone under anaerobic conditions. The enzyme reduced cytochrome c and phenazine methosulfate in the presence of L-galactono-γ-lactone, but not when L-gulono-γ-lactone was used as an electron donor. The K_ms of the enzyme for L-galactono-γ-lactone and cytochrome c were 192 μM and 180 μM, respectively.     

Involvement of Cellular Prion Protein in α-Synuclein Transport in Neurons

The cellular prion protein, encoded by the gene Prnp, has been reported to be a receptor of β-amyloid. Their interaction is mandatory for neurotoxic effects of β-amyloid oligomers. In this study, we aimed to explore whether the cellular prion protein participates in the spreading of α-synuclein. Results demonstrate that Prnp expression is not mandatory for α-synuclein spreading. However, although the pathological spreading of α-synuclein can take place in the absence of Prnp, α-synuclein expanded faster in PrP^C-overexpressing mice. In addition, α-synuclein binds strongly on PrP^C-expressing cells, suggesting a role in modulating the effect of α-synuclein fibrils.     

The Nuclear Localization of Glycogen Synthase Kinase 3β Is Required Its Putative PY-Nuclear Localization Sequences

Glycogen synthase kinase-3β (GSK-3β), which is a member of the serine/threonine kinase family, has been shown to be crucial for cellular survival, differentiation, and metabolism. Here, we present evidence that GSK-3β is associated with the karyopherin β2 (Kap β2) (102-kDa), which functions as a substrate for transportation into the nucleus. A potential PY-NLS motif (¹⁰⁹IVRLRYFFY¹¹⁷) was observed, which is similar with the consensus PY NLS motif (R/K/H)X_2–5PY in the GSK-3β catalytic domain. Using a pull down approach, we observed that GSK-3β physically interacts with Kap β2 both in vivo and in vitro. Secondly, GSK-3β and Kap β2 were shown to be co-localized by confocal microscopy. The localization of GSK-3β to the nuclear region was disrupted by putative Kap β2 binding site mutation. Furthermore, in transient transfection assays, the Kap β2 binding site mutant induced a substantial reduction in the in vivo serine/threonine phosphorylation of GSK-3β, where- as the GSK-3β wild type did not. Thus, our observations indicated that Kap β2 imports GSK-3β through its putative PY NLS motif from the cytoplasm to the nucleus and increases its kinase activity.     

Some Properties and Subcellular Localization of l-Gulono-γ-lactone Dehydrogenase in Euglena gracilis z

The cellulose-binding ability of Geotrichum sp. M111 cells was investigated by the micro-tube method which gives an indication of the binding ability of M111 cells. The optimum pH value and temperature were 3-7 and below 50°C, respectively, from measurement of the aggregation height for a mixture of cellulose powder and M111 cells. The binding constant of 0.3% for M111 cells to cellulose powder was obtained in a 20 mM citrate buffer of pH 5.0 at 30°C. Aggregation was inhibited by such surfactants as sodium dodecylsulfate. The binding ability of M111 cells to cellulose fiber disappeared after a treatment with Driselase or Pronase E. This suggests that the binding ability might be related to the cell surface proteins. The dehydration rate of the distilled waste of sweet potato shouchu was accelerated by the addition of M111 cells. The analysis of dehydration by a linear viscoelastic model suggests that the acceleration effect might have been due to the space increase between cellulose fibers with the cell addition.     

Putative implication of 3′-terminal segment of 18S rRNA in translation initiation of uncapped mRNAs in plants

A putative implication 3′-terminal 18S rRNA segment in the cap-independent initiation of translation on plant ribosomes was studied. It was shown that 3′-terminal segment (nucleotides 1777–1811) of 18S rRNA including the last hairpin 45 was accessible for complementary interactions within 40S ribosomal subunits. Oligonucleotides complementary to this segment of rRNA, when added to wheat germ cell-free protein synthesizing system, specifically inhibited translation of uncapped reporter mRNA encoding β-glucuronidase. In the 5′-untranslated region (UTR), the reporter mRNA contained a leader sequence of potato virus Y (PVY) genomic RNA with fragments complementary to the region 1777–1811. A sequence corresponding to nucleotides 291–316 of PVY, which was complementary to most of the 3′-terminal 18S rRNA segment 1777–1808, was shown to enhance translational efficiency of the reporter mRNAs when placed into 5′-UTR. The obtained results suggest that complementary interactions between 5′-UTR of mRNA and 3′-terminal segment of 18S rRNA can take place during cap-independent translation initiation.