Proximity of the poly(A)-binding protein to a premature termination codon inhibits mammalian nonsense-mediated mRNA decay

mRNA surveillance pathways selectively clear defective mRNAs from the cell. As such, these pathways serve as important modifiers of genetic disorders. Nonsense-mediated decay (NMD), the most intensively studied surveillance pathway, recognizes mRNAs with premature termination codons (PTCs). In mammalian systems the location of a PTC more than 50 nucleotides 5' to the terminal exon-exon junction is a critical determinant of NMD. However, mRNAs with nonsense codons that fulfill this requirement but are located very early in the open reading frame can effectively evade NMD. The unexpected resistance of such mRNAs with AUG-proximal PTCs to accelerated decay suggests that important determinants of NMD remain to be identified. Here, we report that an NMD-sensitive mRNA can be stabilized by artificially tethering the cytoplasmic poly(A) binding protein 1, PABPC1, at a PTC-proximal position. Remarkably, the data further suggest that NMD of an mRNA with an AUG-proximal PTC can also be repressed by PABPC1, which might be brought into proximity with the PTC during cap-dependent translation and 43S scanning. These results reveal a novel parameter of NMD in mammalian cells that can account for the stability of mRNAs with AUG-proximal PTCs. These findings serve to expand current mechanistic models of NMD and mRNA translation.     

A case of acute myeloid leukemia-M2 with trisomy 4 in addition to t(8;21)

t(8;21)(q22;q22) is the most frequently observed karyotypic abnormality associated with acute myeloid leukemia (AML), specifically in FAB-M2. Short-term unstimulated bone marrow (BM) and peripheral blood lymphocyte culture showed 47,XX, +4,t(8;21) in all metaphase plates; and interphase and metaphase results of AML-ETO fusion was positive and trisomy of 4 was confirmed with WCP probes. Trisomy 4 in AML with t(8;21) is a rare numerical abnormality. Here we present such case of patient which may constitute a distinctive subtype.     

The eukaryotic polypeptide chain releasing factor (eRF3/GSPT) carrying the translation termination signal to the 3'-Poly(A) tail of mRNA. Direct association of erf3/GSPT with polyadenylate-binding protein.

The mammalian GTP-binding protein GSPT, whose carboxyl-terminal sequence is homologous to the eukaryotic elongation factor EF1alpha, binds to the polypeptide chain releasing factor eRF1 to function as eRF3 in the translation termination. The amino-terminal domain of GSPT was, however, not required for the binding. Search for other GSPT-binding proteins in yeast two-hybrid screening system resulted in the identification of a cDNA encoding polyadenylate-binding protein (PABP), whose amino terminus is associating with the poly(A) tail of mRNAs presumably for their stabilization. The interaction appeared to be mediated through the carboxyl-terminal domain of PABP and the amino-terminal region of GSPT. Interestingly, multimerization of PABP with poly(A), which is ascribed to the action of its carboxyl-terminal domain, was completely inhibited by the interaction with the amino-terminal domain of GSPT. These results indicate that GSPT/eRF3 may play important roles not only in the termination of protein synthesis but also in the regulation of mRNA stability. Thus, the present study is the first report showing that GSPT/eRF3 carries the translation termination signal to 3'-poly(A) tail ubiquitously present in eukaryotic mRNAs.     

Poly(A)-binding protein facilitates translation of an uncapped/nonpolyadenylated viral RNA by binding to the 3' untranslated region

Viruses employ an alternative translation mechanism to exploit cellular resources at the expense of host mRNAs and to allow preferential translation. Plant RNA viruses often lack both a 5' cap and a 3' poly(A) tail in their genomic RNAs. Instead, cap-independent translation enhancer elements (CITEs) located in the 3' untranslated region (UTR) mediate their translation. Although eukaryotic translation initiation factors (eIFs) or ribosomes have been shown to bind to the 3'CITEs, our knowledge is still limited for the mechanism, especially for cellular factors. Here, we searched for cellular factors that stimulate the 3'CITE-mediated translation of Red clover necrotic mosaic virus (RCNMV) RNA1 using RNA aptamer-based one-step affinity chromatography, followed by mass spectrometry analysis. We identified the poly(A)-binding protein (PABP) as one of the key players in the 3'CITE-mediated translation of RCNMV RNA1. We found that PABP binds to an A-rich sequence (ARS) in the viral 3' UTR. The ARS is conserved among dianthoviruses. Mutagenesis and a tethering assay revealed that the PABP-ARS interaction stimulates 3'CITE-mediated translation of RCNMV RNA1. We also found that both the ARS and 3'CITE are important for the recruitment of the plant eIF4F and eIFiso4F factors to the 3' UTR and of the 40S ribosomal subunit to the viral mRNA. Our results suggest that dianthoviruses have evolved the ARS and 3'CITE as substitutes for the 3' poly(A) tail and the 5' cap of eukaryotic mRNAs for the efficient recruitment of eIFs, PABP, and ribosomes to the uncapped/nonpolyadenylated viral mRNA.     

Enzymatic synthesis of oligonucleotides of defined sequence. The "single addition" of 2(3)-O-dihydrocinnamoyl-nucleoside 5'-diphosphate to a primer oligonucleotide catalyzed by a thermophilic polynucleotide phosphorylase.

Several oligonucleotides of defined sequence were synthesized using 2'(3')-O-dihydrocinnamoyl-nucleoside 5'-diphosphates (DHC-NDP) as substrates for polynucleotide phosphorylase [EC 2.7.7.8] from Thermus thermophilus. The enzyme catalyzed the transfer of one nucleotidyl residue from each of the 2'(3')-O-dihydrocinnamoyl esters of CDP, UDP, and GDP to the 3'-terminus of the primer triadenosine diphosphate, (Ap)2A. The products were shown to be (Ap)3C, (Ap)3U, and (Ap)3G by enzymatic analysis.     

An evolutionarily conserved interaction of tumor suppressor protein Pdcd4 with the poly(A)-binding protein contributes to translation suppression by Pdcd4

The tumor suppressor protein programmed cell death 4 (Pdcd4) has been implicated in the translational regulation of specific mRNAs, however, the identities of the natural Pdcd4 target mRNAs and the mechanisms by which Pdcd4 affects their translation are not well understood. Pdcd4 binds to the eukaryotic translation initiation factor eIF4A and inhibits its helicase activity, which has suggested that Pdcd4 suppresses translation initiation of mRNAs containing structured 5′-untranslated regions. Recent work has revealed a second inhibitory mechanism, which is eIF4A-independent and involves direct RNA-binding of Pdcd4 to the target mRNAs. We have now identified the poly(A)-binding protein (PABP) as a novel direct interaction partner of Pdcd4. The ability to interact with PABP is shared between human and Drosophila Pdcd4, indicating that it has been highly conserved during evolution. Mutants of Pdcd4 that have lost the ability to interact with PABP fail to stably associate with ribosomal complexes in sucrose density gradients and to suppress translation, as exemplified by c-myb mRNA. Overall, our work identifies PABP as a novel functionally relevant Pdcd4 interaction partner that contributes to the regulation of translation by Pdcd4.     

A case of infant swapping by wild northern muriquis (<Emphasis Type="Italic">Brachyteles hypoxanthus</Emphasis>)

Allo-parenting has been observed in a variety of female primates, and typically infants are reunited with their biological mothers assuming that their mothers are alive. We observed an exception to this pattern when two wild northern muriquis (Brachyteles hypoxanthus) exchanged infants of different sexes and then reared their adopted infants through weaning. The process of this exchange began when the infants were 4 and 8 days old, respectively. The mother of a 4-day old female carried and nursed her own daughter and the 8-day old son of a second female. The exchange ended when the second mother was first observed carrying the wrong infant 1.5 days later. This observation raises questions about the age and mechanisms of mother–infant recognition in this species, and about assumptions of mother–infant relatedness based on behavioral observations alone.     

Detoxification of cholera toxin without removal of its immunoadjuvanticity by the addition of (STa-related) peptides to the catalytic subunit. A potential new strategy to generate immunostimulants for vaccination

Peptides related to the heat-stable enterotoxin STa were fused to the N terminus of the A-subunit of cholera toxin (CTA) to explore whether peptide additions could help generate detoxified cholera toxin (CT) derivatives. Proteins carrying APRPGP (6-CTA), ASRCAELCCNPACPAP (16-CTA), or ANSSNYCCELCCNPACTGCYPGP (23-CTA) were genetically constructed. Using a two-plasmid system these derivatives were co-expressed in Vibrio cholerae with cholera toxin B-subunit (CTB) to allow formation and secretion of holotoxin-like molecules (engineered CT, eCTs). Purified eCTs maintained all normal CT properties yet they were more than 10-fold (eCT-6), 100-fold (eCT-16), or 1000-fold (eCT-23) less enterotoxic than wild-type CT. The inverse correlation between enterotoxicity and peptide length indicated sterical interference with the ADP-ribosylating active site in CTA. This interpretation agreed with greater than 1000-fold reductions in cAMP induction, with reductions, albeit not proportional, in in vitro agmatine ADP-ribosylation, and was supported by molecular simulations. Intranasal immunization of mice demonstrated that eCTs retained their inherent immunogenicity and ability to potentiate immune responses to a co-administered heterologous protein antigen, although in variable degrees. Therefore, the addition of STa-related peptides to CTA reduced the toxicity of CT while partly preserving its natural immunoadjuvanticity. These results suggest peptide extensions to CTA are a useful alternative to site-directed mutagenesis to detoxify CT. The simplicity of the procedure, combined with efficient expression and assembly of derivatives, suggests this approach could allow for large scale production of detoxified, yet immunologically active CT molecules.     

Negative effect of chromosome 1A on dough strength shown by modification of 1D addition in durum wheat (<Emphasis Type="Italic">Triticum durum</Emphasis>)

A monosomic addition line of Aegilops tauschii chromosome 1D in Triticum durum cv. PBW114 was produced in 1990. This line was self-pollinated and maintained for several generations while following the presence of chromosome 1D carrying the gene for red glume color. Cytological analysis indicated that two of the three derivative lines had substitution of chromosome 1D for 1A and another had substitution of chromosome 1D for 1B. One of these lines carried a pair of small chromosomes in addition to the 1D chromosome. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the derived lines showed the presence of high-molecular-weight (HMW) glutenin encoded by the Glu-D1 locus. The small chromosome found in one of the lines had nearly regular pairing and transmission to daughter nuclei. Fluorescent in situ hybridization (FISH) and analysis of molecular markers indicated that the small chromosome was derived from the short arm of chromosome 1A and carried the Glu-A3 locus. Microsatellite mapping based on the deletion bin map revealed that the small chromosome had terminal deletions on both the terminal and centromeric sides. The line with the small chromosome showed improvement of the sodium dodecyl sulfate (SDS)-sedimentation value as compared to parent durum. However, the increase in SDS-sedimentation value was more significant in the substitution line of chromosome 1D for 1A without the small chromosome. These facts suggest a negative effect of the Glu-A3 locus on dough strength. The sequence of the Glu-D1 locus from these lines showed that the HMW glutenin subunits were Ae. tauschii specific 2^t + T2, which were previously found to be associated with poor rheological properties and bread loaf volume in synthetic hexaploid wheat by other workers. Thus, the significant improvement in the SDS-sedimentation value of the substitution line of 1D for 1A suggests that the absence of the negative effect of chromosome 1A on quality is more important than the presence of Glu-D1 of Ae. tauschii.     

Transfer of resistance against the beet cyst nematode from radish (<Emphasis Type="Italic">Raphanus sativus</Emphasis>) to rape (<Emphasis Type="Italic">Brassica napus</Emphasis>) by monosomic chromosome addition

In rape (Brassica napus), no resistance to the beet cyst nematode (BCN) Heterodera schachtii is available. This study was carried out to determine the specific chromosome(s) of resistant radish (Raphanus sativus) carrying the gene(s) for nematode resistance as a prequisite to convert rape from a host into a trap crop for this pest. A Raphanobrassica progeny of 25 plants was analyzed which segregated for all nine chromosomes of the Raphanus genome in a genetic background of synthetic rape. The number of radish chromosomes was determined by fluorescence in situ hybridization, using the Raphanus-specific DNA probe pURsN; and their type was identified by chromosome-specific randomly amplified polymorphic DNA markers. Five different multiple rape–radish chromosome additions (comprising the whole set of nine radish chromosomes, a–i) were selected and crossed to rape. For each cross-progeny, the number of cysts on plant roots was counted 42 days after inoculation with a L2 larvae suspension. Simultaneously, the plants were characterized for the presence or absence of individual radish chromosomes, using sets of chromosome-specific markers. Thus, the effect of each radish chromosome on cyst number was tested. Chromosome d had a major resistance effect, whereas the presence/absence of the other radish chromosomes had nearly no influence on cyst number. Plants with added chromosome d showed a resistance level comparable with that of the radish donor parent. The analysis in the cross to rape of a plant monosomic only for chromosome d confirmed the strong effect of this chromosome on nematode resistance. A further experiment comprising seven crosses using winter rape breeding lines and monosomic addition line d as pollen parent provided the same results on a broader genetic basis. In each case, the added chromosome d in a single dosage caused nearly the full resistance of the radish donor. Resistance was independent of the glucosinolate content in the roots. The possibilities for stabilizing BCN resistance in rape and its use for other crops and nematodes are discussed.Communicated by C. Möllers     

Damage and repair in mammalian cells after exposure to non-ionizing radiations. II. Photoreactivation and killing of rat kangaroo cells (Potorous tridactylus) and Herpes simplex virus-1 by exposure to fluorescent "white" light or sunlight

Photoreactivation (PR) of ultraviolet (254 nm)-inactivated cornea cells of the potoroo (or rat kangaroo; Potorous tridacylus) has been studied at wavelengths greater than 375 nm from either fluorescent "white" light or sunlight. In both cases the PR kinetics curves pass through maxima, which most likely result from the superposition of concomitant inactivation by the photoreactivating light. The inactivating effect of light was directly demonstrated for non-UV-irradiated cells, permitting correction of the PR curves. Wavelengths greater than 475 nm, and even greater than 560 nm, which do not noticeably damage cells, still photoreactivate, though less effectively than shorter wavelengths. Light treatment of UV-inactivated Herpes simplex Virus-1 (HSV-1) after infection leads to PR effects resembling those observed for cells, while light treatment of unirradiated virus after infection likewise causes inactivation. The "fluence-reduction factor" of PR, which is greater than 3 for the virus, exceeds that for the cells, where it decreases with increasing UV fluence. In vitro tests have indicated that sunlight greater than 375 nm causes photorepairable DNA lesions which are virtually fully repaired by the same light. Thus cell inactivation resulting from these solar wavelengths must be due to non-photorepairable damage.     

A gene expression analysis of syncytia laser microdissected from the roots of the <Emphasis Type="Italic">Glycine max</Emphasis> (soybean) genotype PI 548402 (Peking) undergoing a resistant reaction after infection by <Emphasis Type="Italic">Heterodera glycines</Emphasis> (soybean cyst nematode)

The syncytium is a nurse cell formed within the roots of Glycine max by the plant parasitic nematode Heterodera glycines. Its development and maintenance are essential for nematode survival. The syncytium appears to undergo two developmental phases during its maturation into a functional nurse cell. The first phase is a parasitism phase where the nematode establishes the molecular circuitry that during the second phase ensures a compatible interaction with the plant cell. The cytological features of syncytia undergoing susceptible or resistant reactions appear the same during the parasitism phase. Depending on the outcome of any defense response, the second phase is a period of syncytium maintenance (susceptible reaction) or failure (resistant reaction). In the analyses presented here, the localized gene expression occurring at the syncytium during the resistant reaction was studied. This was accomplished by isolating syncytial cells from Glycine max genotype Peking (PI 548402) by laser capture microdissection. Microarray analyses using the Affymetrix^® soybean GeneChip^® directly compared Peking syncytia undergoing a resistant reaction to those undergoing a susceptible reaction during the parasitism phase of the resistant reaction. Those analyses revealed lipoxygenase-9 and lipoxygenase-4 as the most highly induced genes in the resistant reaction. The analysis also identified induced levels of components of the phenylpropanoid pathway. These genes included phenylalanine ammonia lyase, chalcone isomerase, isoflavone reductase, cinnamoyl-CoA reductase and caffeic acid O-methyltransferase. The presence of induced levels of these genes implies the importance of jasmonic acid and phenylpropanoid signaling pathways locally at the site of the syncytium during the resistance phase of the resistant reaction. The analysis also identified highly induced levels of four S-adenosylmethionine synthetase genes, the EARLY-RESPONSIVE TO DEHYDRATION 2 gene and the 14-3-3 gene known as GENERAL REGULATORY FACTOR 2. Subsequent analyses studied microdissected syncytial cells at 3, 6 and 9 days post infection (dpi) during the course of the resistant reaction, resulting in the identification of signature gene expression profiles at each time point in a single G. max genotype, Peking.     

A case study of the past CH4 cycle in lakes by the combined use of dual isotopes (carbon and hydrogen) and ancient DNA of methane-oxidizing bacteria: rearing experiment and application to Lake Remoray (eastern France)

Aquatic Ecology - This study aims at estimating the potential of the hydrogen stable isotope (δ2H) analysis of chironomid remains (HC) to reconstruct past changes in the methane (CH4) cycle in...     

Determination of ochratoxin A in tissues of wild boar (<Emphasis Type="Italic">Sus scrofa</Emphasis> L.) by enzymatic digestion (ED) coupled to high-performance liquid chromatography with a fluorescence detector (HPLC-FLD)

Ochratoxin A (OTA) is a secondary toxic metabolite synthesized by Aspergillus or Penicillium species, which can contaminate various crops. The International Agency for Research on Cancer (IARC) classified OTA as a group 2B possible human carcinogen. The aim of the present study was to assess OTA concentrations in tissues of wild boar (Sus scrofa L.) from Tuscany (Italy). Over a period of 2 years, samples of muscle, liver, and kidney from 48 wild boars were collected and concentrations of OTA were determined by enzymatic digestion (ED) coupled to high-performance liquid chromatography with a fluorescence detector (HPLC-FLD). The highest concentrations of OTA were found in the kidneys of the 48 wild boars analyzed. No difference in concentrations was found based on years of collection and sex while a significantly higher OTA concentration was found in the kidney of the young wild boars with respect to the adult one. Monitoring the quality of meat destined for transformation is a priority in order to decrease the possibility of toxin carry-over to humans. The present study showed that contamination of wild boar meat products by OTA represents a potential emerging source of OTA.     

Kinetics of reaction of nitrite with deoxy hemoglobin after rapid deoxygenation or predeoxygenation by dithionite measured in solution and bound to the cytoplasmic domain of band 3 (SLC4A1)

The reaction of deoxyhemoglobin with nitrite was characterized in the presence of dithionite using hemoglobin in solution or bound to the cytoplasmic domain of band 3 (CDB3). Deoxyhemoglobin was generated by predeoxygenation (nitrogen flushing followed by addition of dithionite), or transiently, by rapidly mixing oxyhemoglobin with nitrite and dithionite simultaneously. Wavelength-dependent kinetic studies confirmed the formation of nitrosyl hemoglobin. Furthermore, the rate of reaction was independent of dithionite concentration, indicating that dithionite does not reduce nitrite to nitric oxide directly. Model simulation studies showed that superoxide anion generated by dithionite reduction of molecular oxygen was not a factor in the reaction kinetics. CDB3-bound hemoglobin reacted faster with nitrite than did hemoglobin in solution. This difference was most pronounced for predeoxygenated hemoglobin and least pronounced for rapidly deoxygenated hemoglobin. The smaller difference observed in the rapid deoxygenation experiment was associated with much faster kinetics compared to the predeoxygenation experiment. Model simulation studies showed, and literature evidence indicates, that faster kinetics in the rapid deoxygenation experiment were related to the initial presence of R-state Hb(II)O 2 alphabeta dimers, both in dilute solution and when bound to CDB3. Thus, rapidly deoxygenated CDB3-bound hemoglobin alphabeta dimers react 5-fold faster with nitrite than predeoxygenated tetrameric hemoglobin in solution. Faster nitrite reductase kinetics for CDB3-bound hemoglobin suggests the possibility of preferential nitric oxide generation at the inner surface of the erythrocyte membrane, thus coupling the release of oxygen from hemoglobin to the production and successful release of nitric oxide from the erythrocyte, and the regulation of blood flow.     

A questionnaire survey regarding the support needed by <Emphasis Type="Italic">Yogo</Emphasis> teachers to take care of students suspected of having eating disorders (second report)

Background

The lowering of the age of onset and chronicity have been key problems related to eating disorders (EDs). As the proportion of teens in the estimated onset ages has increased, it has become important to detect students with EDs and to clarify how they can be supported. Though epidemiological surveys of Yogo teachers (school nurse/health science teachers) have been conducted to inquire about the number of such students, none of these were done according to ED type based on DSM-5. Thus, we conducted a wide area survey in Japan with the goal of proposing a better framework of support for Yogo teachers in their efforts to care for students with EDs.

Methods

A questionnaire survey organized by ED type (based on DSM-5) was administered to Yogo teachers working at elementary/junior high/senior high/special needs schools in four prefectures of Japan in 2015, and 1,886 responses were obtained. Based on the results, the encounter rates (the proportion of Yogo teachers who had encountered a student with an ED) were calculated, and factors that could affect the rates were examined by logistic regression analysis.

Results

The order of the encounter rates of the ED types was as follows: Anorexia Nervosa (AN)?>?Bulimia Nervosa (BN)?>?Avoidant/Restrictive Food Intake Disorder (ARFID)?>?Binge Eating Disorder (BED)?>?Others. The factors significantly affecting the rates were “location, school type, number of students, experience years, and AN knowledge” for AN, “school type, experience years, and BN knowledge” for BN, “school type, experience years, and BED knowledge” for BED, “location, experience years, and ARFID knowledge” for ARFID, and “school type, experience years, and Others knowledge” for Others.

Conclusions

Because the encounter rate of AN was the highest, providing support for AN would be the most effective. Moreover, one factor that affected the encounter rate of all ED types was ED knowledge. In addition to this, senior high schools had the highest encounter rates for AN, BN and BED, and special needs schools had the highest rates for Others. These findings imply that, in order to detect and support ED students at an early stage, it is necessary to offer knowledge of the most prevalent ED types to Yogo teachers at the corresponding school type.