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La cellule est en permanence sujette à des attaques et soumise à des évènements stressants. D’un point de vue évolutif, l’organisme doit s’adapter à son environnement et faire face aux atteintes dont il peut être victime. Pour survivre, celui-ci doit répondre aux dommages reçus de manière la plus optimale possible tout en limitant l’occurrence de mutations pouvant mener à l’apparition de cancer. Afin d’y parvenir, l’organisme a développé les suppresseurs de tumeurs, gérant des voies de signalisation cellulaire complexes, limitant l’apparition de mutations à l’ADN, stimulant sa réparation et, en dernier recours, activant l’arrêt permanent du cycle cellulaire, la sénescence, ou la mort cellulaire, l’apoptose. Le défaut de fonctionnement de ces mécanismes entraîne l’accumulation de mutations favorisant l’initiation et la progression de la tumorigenèse. Les erreurs traductionnelles, en contribuant à l’expansion du protéome, ont elles aussi été reconnues comme faisant partie d’un mécanisme pouvant être utilisé pour répondre au stress de sélection. Cependant, chez les organismes plus développés ainsi que dans la tumorigenèse, leur rôle reste peu compris à ce jour. En revanche, si ce phénomène peut contribuer à l’oncogenèse, les mécanismes suppresseurs de tumeurs devraient, en théorie, les prévenir. Les travaux présentés dans ce mémoire ont été entrepris dans le but d’étudier les erreurs traductionnelles survenant à la terminaison, la translecture du codon d’arrêt (TR), en contexte de sénescence. En utilisant une construction double luciférase séparée par des contextes de terminaison normale ou de TR programmé, nos résultats montrent que l’induction de la sénescence par l’activation d’oncogène, l’activation de suppresseurs de tumeurs et des agents chimiothérapeutiques, est associée avec une diminution significative du TR. De surcroît, nous avons observé que les cellules échappant à la sénescence montrent un niveau de TR augmenté. Nous avons également démontré ces effets en utilisant AGO1x un isoforme dépendant du TR impliqué dans la progression du cancer du sein. D’autre part, nos travaux ont visé à établir un lien mécanistique entre les suppresseurs de tumeurs impliqués dans la sénescence et la régulation du TR. Dans un premier temps, en déplétant p53 et RB nous avons montré une régulation majoritairement médiée par la voie RB. Les résultats ont été confirmés en ciblant de diverses manières la voie RB. Quant à l'implication de p53, nous II avons montré en utilisant des cellules p53-null que la réduction de TR dans la sénescence était indépendante de cette voie. Les données présentées dans ce mémoire contribuent à une meilleure compréhension des mécanismes par lesquels les voies de signalisation des suppresseurs de tumeurs pourraient limiter la carcinogenèse. Ainsi, à la lumière de nos observations, nous proposons que l’amélioration de l’efficacité de terminaison observée en sénescence puisse être un nouveau mécanisme antitumoral médié par la voie RB., The cell is constantly victim of attacks and stressful events. From an evolutionary perspective, the organism must be able to adapt to its environment and respond to the harm it may suffer. To survive, it must respond to damage received in the most optimal way possible, by limiting the appearance of mutations that can lead to cancer. To achieve this, tumor suppressors evolved, managing complex cellular signaling pathways limiting DNA mutations, stimulating DNA repair and, in the worst case, activating a permanent cell cycle arrest, namely senescence, or cell death(apoptosis). Defects in these mechanisms may lead to the accumulation of mutations favoring the initiation of tumorigenesis. Translational errors, by contributing to the expansion of cellular proteome, have also been associated as a mechanism that can be used to respond to selection stress. However, their role in more developed organisms, as well as in tumorigenesis, is still poorly understood. On the other hand, if this phenomenon can contribute to oncogenesis, tumor suppressor mechanisms should theoretically prevent them to occur. The work presented in this thesis was undertaken with the aim of studying translational errors occurring at the termination, the translational readthrough (TR), in the context of senescence. Using a dual luciferase construct with normal termination or programmed TR context, our results show that induction of senescence by oncogene activation, activation of tumor suppressors and chemotherapeutic drugs is associated with significant decrease in TR. In addition, we observed that cells escaping senescence show an increased level of TR. We have also demonstrated these effects using AGO1x a TR-dependent isoform implicated in the breast cancer progression. Furthermore, our work aimed to establish a mechanistic link between tumor suppressors involved in senescence and in the regulation of TR. Firstly, by depleting p53 and RB, we showed that the reduction of TR seen in senescence is mainly mediated by the RB pathway. These results were confirmed by targeting the RB pathway in various ways. As for the implication of p53, we showed by using p53-null cells that the reduction of TR in senescence was independent of this pathway. The data presented in this thesis contribute to a better understanding of the mechanisms by which tumor suppressor signaling pathways could limit carcinogenesis. Thus, in light of our IV findings, we propose that the improved termination efficiency observed in senescence may be a novel RB pathway-mediated antitumor mechanism.

We previously reported that ribosome stalling at AUG-stop sequences in the 5'-untranslated region plays a critical role in regulating the expression of Arabidopsis thaliana NIP5;1, which encodes a boron uptake transporter, in response to boron conditions in media. This ribosome stalling is triggered specifically by boric acid, but the mechanisms are unknown. Although upstream open reading frames (uORFs) are known in many cases to regulate translation through peptides encoded by the uORF, AUG-stop stalling does not involve any peptide synthesis. The unique feature of AUG-stops - that termination follows immediately after initiation - suggests a possible effect of boron on the translational process itself. However, the generality of AUG-stop-mediated translational regulation and the effect of boron on translation at the genome scale are not clear. Here, we conducted a ribosome profiling analysis to reveal the genome-wide regulation of translation in response to boron conditions in A. thaliana shoots. We identified hundreds of translationally regulated genes that function in various biological processes. Under high-boron conditions, transcripts with reduced translation efficiency were rich in uORFs, highlighting the importance of uORF-mediated translational regulation. We found 673 uORFs that had more frequent ribosome association. Moreover, transcripts that were translationally downregulated under high-boron conditions were rich in minimum uORFs (AUG-stops), suggesting that AUG-stops play a global role in the boron response. Metagene analysis revealed that boron increased the ribosome occupancy of stop codons, indicating that this element is involved in global translational termination processes.

Dual-specificity tyrosine phosphorylation-regulated kinase 1 A (DYRK1A) is essential for human development, and DYRK1A haploinsufficiency is associated with a recognizable developmental syndrome and variable clinical features. Here, we present a patient with DYRK1A haploinsufficiency syndrome, including facial dysmorphism, delayed motor development, cardiovascular system defects, and brain atrophy. Exome sequencing identified a novel de novo heterozygous mutation of the human DYRK1A gene (c.1185dup), which generated a translational termination codon and resulted in a C-terminally truncated protein (DYRK1A-E396ter). To study the molecular effect of this truncation, we generated mammalian cell and Drosophila models that recapitulated the DYRK1A protein truncation. Analysis of the structure and deformation energy of the mutant protein predicted a reduction in protein stability. Experimentally, the mutant protein was efficiently degraded by the ubiquitin-dependent proteasome pathway and was barely detectable in mammalian cells. More importantly, the mutant kinase was intrinsically inactive and had little negative impact on the wild-type protein. Similarly, the mutant protein had a minimal effect on Drosophila phenotypes, confirming its loss-of-function in vivo. Together, our results suggest that the novel heterozygous mutation of DYRK1A resulted in loss-of-function of the kinase activity of DYRK1A and may contribute to the developmental delay observed in the patient.

PURPOSE: Cystic fibrosis transmembrane conductance regulator (CFTR) and adhesion G protein-coupled receptor G2 (ADGRG2) have been identified as the main pathogenic genes in congenital bilateral absence of the vas deferens (CBAVD), which is an important cause of obstructive azoospermia. This study aimed to identify the disease-causing gene in two brothers with CBAVD from a Chinese consanguineous family and reveal the intracytoplasmic sperm injection (ICSI) outcomes in these patients. METHODS: Whole-exome sequencing and Sanger sequencing were used to identify the candidate pathogenic genes. Real-time polymerase chain reaction, immunohistochemistry, and immunofluorescence were used to assess the expression of the mutant gene. Moreover, the ICSI results from both patients were retrospectively reviewed. RESULTS: A novel hemizygous loss-of-function mutation (c.G118T: p.Glu40*) in ADGRG2 was identified in both patients with CBAVD. This mutation is absent from the human genome databases and causes an early translational termination in the third exon of ADGRG2. Expression analyses showed that both the ADGRG2 mRNA and the corresponding protein were undetectable in the proximal epididymal tissue of ADGRG2-mutated patients. ADGRG2 expression was restricted to the apical membranes of non-ciliated epithelia in human efferent ducts, which was consistent with a previous report in mice. Both ADGRG2-mutated patients had normal spermatogenesis and had successful clinical outcomes following ICSI. CONCLUSIONS: Our study verifies the pathogenic role of ADGRG2 in X-linked CBAVD and broadens the spectrum of ADGRG2 mutations. In addition, we found positive ICSI outcomes in the two ADGRG2-mutated CBAVD patients. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s10815-020-01779-6) contains supplementary material, which is available to authorized users.

Objectives To investigate the genetic causes of hearing loss with enlarged vestibular aqueduct (EVA) in two children from unrelated two Chinese families. Methods Sanger sequencing of all coding exons in SLC26A4 (encoding Pendrin protein) was performed on the two patients, their sibling and parents respectively. To predict and visualize the potential functional outcome of the novel variant, model building, structure analysis, and in silico analysis were further conducted. Results The results showed that the proband from family I harbored a compound heterozygote of SLC26A4 c.1174A>T (p.N392Y) mutation and c.1181delTCT (p.F394del) variant in exon 10, potentially altering Pendrin protein structure. In family II, the proband was identified in compound heterozygosity with a known mutation of c.919-2A>G in the splice site of intron 7 and a novel mutation of c.1023insC in exon 9, which results in a frameshift and translational termination, consequently leading to truncated Pendrin protein. Sequence homology analysis indicated that all the mutations localized at high conservation sites, which emphasized the significance of these mutations on Pendrin spatial organization and function. Conclusion In summary, this study revealed two compound heterozygous mutations (c.1174A>T/c.1181delTCT; c.919- 2A>G/c.1023insC) in Pendrin protein, which might account for the deafness of the two probands clinically diagnosed with EVA. Thus this study contributes to improve understanding of the causes of hearing loss associated with EVA and develop a more scientific screening strategy for deafness.

Supplemental Digital Content is available in the text, Colorectal cancer, especially colon adenocarcinoma (COAD), is associated with significant morbidity and mortality worldwide. Long noncoding RNA (lncRNA) has been implicated in tumorigenesis. The aim of the present study was to elucidate the potential diagnostic and prognostic values of lncRNA FRGCA in COAD. The data of 438 COAD patients were retrieved for analysis. Diagnostic significance was evaluated using tumor and nontumor tissues. Prognostic significance was evaluated using a Cox proportional regression model. Stratified analysis was performed to identify associations between clinical factors and lncRNA FRGCA expression. A nomogram was constructed using the clinical factors and lncRNA FRGCA for survival prediction. Enrichment analysis identified gene ontologies and metabolic pathways of mRNAs with high Pearson correlation coefficients with lncRNA FRGCA. lncRNA FRGCA was highly expressed in tumor tissues of COAD and demonstrated diagnostic value (area under curve = 0.763, P

BackgroundIncreasing evidence has demonstrated the correlation between hepatocellular carcinoma (HCC) prognosis and RNA binding proteins (RBPs) dysregulation. Thus, we aimed to develop and validate a reliable prognostic signature that can estimate the prognosis for HCC.MethodsGene expression profiling and clinical information of 374 HCC patients were derived from the TCGA data portal. The survival-related RBP pairs were determined using univariate cox-regression analysis and the signature was built based on LASSO analysis. All patients were divided patients into high-and low-risk groups according to the optimal cut off of the signature score determined by time-dependent receiver operating characteristic (ROC) curve analysis. The predictive value of the signature was further validated in an independent cohort.ResultsA 37-RBP pairs signature consisting of 61 unique genes was constructed which was significantly associated with the survival. The RBP-related signature accurately predicted the prognosis of HCC patients, and patients in high-risk groups showed poor survival in two cohorts. The novel signature was an independent prognostic factor of HCC in two cohorts (all P ConclusionThe novel proposed RBP-related signature based on relative expression orderings could serve as a promising independent prognostic biomarker for patients with HCC, and could improve the individualized survival prediction in HCC.

In this study, we analyzed the Rc and Rd genes that are responsible for the coloration of rice pericarps from six upland rice varieties. We also examined the association of pericarp coloration to the single nucleotide polymorphism in 9-cis-epoxycarotenoid dioxygenase 2 (NCED2), a key gene involved in abscisic acid (ABA) biosynthesis. Our findings demonstrated that all the upland rice varieties analyzed have a Rd gene which encodes a complete dihydroflavonol-4-reductase without early translational termination codon irrespective of their pericarp colors. However, the upland rice varieties with white pericarps were found to have a defective Rc gene with a 14-base deletion at exon 7 which could disrupt the function of a positive regulator of proanthocyanidin biosynthesis. In addition, the NCED2 genes from the upland rice varieties with white pericarps in this study have a C-allele while the NCED2 genes from Pandasan Red, Tomou and Taragang varieties that bear red pericarps were found to have a T-allele which was reported to be associated with a higher ABA level in upland rice. A better understanding of the gene sequences of upland rice varieties with red pericarp may provide important information for rice breeding programs. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13205-020-2092-y) contains supplementary material, which is available to authorized users.

The ACTN3 gene encodes α-actinin-3 protein, which stabilizes the contractile apparatus at the Z-line in skeletal muscle cell fast fibers. A nonsense mutation of the arginine (R) at the codon for amino acid 577 of the ACTN3 gene generates a premature termination codon (PTC) and produces the R577X polymorphism in humans (X specifies translational termination). The ACTN3 577X genotype abolishes α-actinin-3 protein production due to targeted degradation of the mutant transcript by the cellular nonsense-mediated mRNA decay (NMD) system, which requires mRNA splicing. In humans, α-actinin-3 deficiency can decrease sprinting and power performance as well as skeletal muscle mass and strength. Here we investigated whether suppression of the in-frame PTC induced by treatment with the aminoglycosides gentamicin and G418 that promote termination codon readthrough could allow production of full-length α-actinin-3 protein from ACTN3 577X. We constructed expression plasmids encoding mature mRNA that lacks introns or pre-mRNA, which carries introns for the ACTN3 577X gene (X and Xpre, respectively) and transfected the constructs into HEK293 cells. Similar constructs for the ACTN3 577R gene were used as controls. HEK293 cells carrying the X gene, but not the Xpre gene, expressed exogenous truncated α-actinin-3 protein, indicating NMD-mediated suppression of exogenous Xpre expression. Cells treated with aminoglycosides produced exogenous full-length α-actinin-3 protein in X-transfected cells, but not in Xpre-transfected cells. The NMD inhibitor caffeine prevented suppression of Xpre expression and thereby induced production of full-length α-actinin-3 protein in the presence of aminoglycoside. Together these results indicate that the ACTN3 R577X polymorphism could be a novel target for readthrough therapy, which may affect athletic and muscle performance in humans.

When a stop codon is at the 80S ribosomal A site, there are six nucleotides (+4 to +9) downstream that are inferred to be occupying the mRNA channel. We examined the influence of these downstream nucleotides on translation termination success or failure in mammalian cells at the three stop codons. The expected hierarchy in the intrinsic fidelity of the stop codons (UAA>UAG>>UGA) was observed, with highly influential effects on termination readthrough mediated by nucleotides at position +4 and position +8. A more complex influence was observed from the nucleotides at positions +5 and +6. The weakest termination contexts were most affected by increases or decreases in the concentration of the decoding release factor (eRF1), indicating that eRF1 binding to these signals was rate-limiting. When termination efficiency was significantly reduced by cognate suppressor tRNAs, the observed influence of downstream nucleotides was maintained. There was a positive correlation between experimentally measured signal strength and frequency of the signal in eukaryotic genomes, particularly in Saccharomyces cerevisiae and Drosophila melanogaster. We propose that termination efficiency is not only influenced by interrogation of the stop signal directly by the release factor, but also by downstream ribosomal interactions with the mRNA nucleotides in the entry channel.

Objective: To identify the region conferring stability to pBSSB2 (a linear plasmid, pBSSB1, containing a kanamycin cassette), which is unique to Indonesian isolates of Salmonella enterica serovar Typhi. Methods: The open reading frame (ORF) 009 was identified as a toxin coding gene in the plasmid through introduction of translational termination codons in the ORF. Results: The stability function was located in a fragment that spanned nucleotides 5 766 to 6 828 in the linear plasmid genetic map. Ectopic expression of ORF009 in pBAD18 vector indicated ORF009 codes for a toxin. This fragment could stabilize plasmid pUC18 previously destabilized through mutation of the pcnB (plasmid copy number control) gene that codes for polyA polymerase. Majority of the cells expressing ORF009 were non-viable according to phase contrast microscopy. Conclusions: This study demonstrated that a linear plasmid fragment that carries a gene encoding a toxin possibly conferred stability to the parent plasmid. It was able to stabilize a multicopy plasmid of Escherichia coli.

Hajdu-Cheney syndrome (HCS) is a rare autosomal-dominant disorder primarily characterized by acro-osteolysis and early-onset osteoporosis. Genetically, HCS is caused by nonsense or deletion mutations within exon 34 of the NOTCH2 gene, resulting in premature translational termination and production of C-terminally truncated NOTCH2 proteins that are predicted to activate NOTCH2-dependent signaling. To understand the role of Notch2 in bone remodeling, we developed a mouse model of HCS by introducing a pathogenic mutation (6272delT) into the murine Notch2 gene. By μCT and undecalcified histology, we observed generalized osteopenia in two independent mouse lines derived by injection of different targeted embryonic stem (ES) cell clones, yet acro-osteolysis did not occur until the age of 52 weeks. Cellular and dynamic histomorphometry revealed a high bone turnover situation in Notch2+/HCS mice, since osteoblast and osteoclast indices were significantly increased compared with wild-type littermates. Whereas ex vivo cultures failed to uncover cell-autonomous gain-of-functions within the osteoclast or osteoblast lineage, an unbiased RNA sequencing approach identified Tnfsf11 and Il6 as Notch-signaling target genes in bone marrow cells cultured under osteogenic conditions. Because we further observed that the high-turnover pathology of Notch2+/HCS mice was fully normalized by alendronate treatment, our results demonstrate that mutational activation of Notch2 does not directly control osteoblast activity but favors a pro-osteoclastic gene expression pattern, which in turn triggers high bone turnover. © 2017 American Society for Bone and Mineral Research.

Endosperm development plays an important role in the determination of grain weight in maize. In this context, a spontaneous recessive shrunken kernel mutant, shrunken1-m (sh1-m), identified from improved maize inbred line Zheng58 (Gai-Z58) was studied. Physiological and microscopic analysis revealed that the sh1-m mutant significantly increased the content of amylose, and its starch granules showed distinctive morphology. Utilizing 1877 recessive sh1-m individuals from a BC1 segregating population of B73/sh1-m and 19 molecular markers, the sh1-m gene was limited to a 2.1 kb interval within the GRMZM2G089713 gene. A 5-bp substitution in sh1-m covering the 3′ end of the thirteenth exon and the 5′ splicing site of the thirteenth intron led to a missense mutation and a mis-splicing site that resulted in early translational termination. sh1 reference mutant sh1-912A (shrunken-912A) had a single transversion at the 3′ splice junction of the second intron resulting in a mis-splicing site causing the 13-bp spliced out in the third exon and the initiation codon to shift to 118-bp downstream. Further genetic allelism tests between them confirmed that sh1-m is a novel allele of the Sh1 locus. Sh1 was constitutively expressed in all tested tissues, and its expression pattern/level was not significantly changed in developing seeds of the sh1-m mutant. Transcript mRNA patterns/levels of some other genes involved in starch biosynthesis was significantly altered in the sh1-m mutant. These results provide direct evidence that sh1-m plays a key role in starch biosynthesis and provide valuable information for grain quality research and breeding in maize.

Background Leukocyte adhesion deficiency (LAD) or CD18 deficiency is an autosomal recessive immunodeficiency which has been described in people, cattle, dogs, and knockout mice. Objectives The study goals were to characterize the clinicopathologic, immunologic, and molecular genetic features of feline LAD (FLAD) in a neutered male adult Domestic Longhair cat with severe leukocytosis and recurrent infections. Methods Flow cytometry evaluated surface expression of CD18 on neutrophils. In vitro functional assays assessed CD18-dependent neutrophil adhesion and T-cell proliferation. Genomic DNA and cDNA were used to identify a causative mutation in the coding sequence of the integrin β2 subunit (ITGB2) gene. Results The affected cat developed periodontitis during the first months of life followed by recurrent infections poorly responsive to antibiotic therapy, accompanied by extreme neutrophilia. Neutrophils from the proband, compared to feline controls, did not express any CD18 on the cell surface. Adhesion of affected neutrophils was severely impaired with and without phorbol-myristate-acetate activation. The proband's T-cells proliferated weakly to 1 pg but normally to 100 pg staphylococcal enterotoxin A, suggesting a CD18-independent T-cell response at higher doses. Molecular genetic analysis of the ITGB2 gene revealed a 24 bp deletion at the exon 2 to intron 2 boundary (c.46_58 + 11del), predicting premature translational termination due to abnormal splicing of exon 1 to exon 3 or 4. Conclusions Feline LAD exhibits features similar to LAD in other species. However, clinical episodes in FLAD appeared milder allowing for an extended life expectancy under long-term antimicrobial therapy, possibly due to an alternative, CD18-independent T-cell proliferation pathway.

Selenoproteins contain the amino acid selenocysteine (Sec), co-translationally inserted at a predefined UGA opal codon by means of Sec-specific translation machineries. In Escherichia coli, this process is dependent upon binding of the Sec-dedicated elongation factor SelB to a Sec insertion sequence (SECIS) element in the selenoprotein-encoding mRNA and competes with UGA-directed translational termination. Here, we found that Sec can also be efficiently incorporated at a predefined UAG amber codon, thereby competing with RF1 rather than RF2. Subsequently, utilizing the RF1-depleted E. coli strain C321.ΔA, we could produce mammalian selenoprotein thioredoxin reductases with unsurpassed purity and yield. We also found that a SECIS element was no longer absolutely required in such a system. Human glutathione peroxidase 1 could thereby also be produced, and we could confirm a previously proposed catalytic tetrad in this selenoprotein. We believe that the versatility of this new UAG-directed production methodology should enable many further studies of diverse selenoproteins.

SUMMARYMitochondrial dysfunction and proteostasis failure frequently coexist as hallmarks of neurodegenerative disease. How these pathologies are related is not well understood. Here we describe a phenomenon termed MISTERMINATE (mitochondrial stress-induced translational termination impairment and protein carboxyl terminal extension), which mechanistically links mitochondrial dysfunction with proteostasis failure. We show that mitochondrial dysfunction impairs translational termination of nuclear-encoded mitochondrial mRNAs including complex-I 30kD subunit (C-I30) mRNA, occurring on mitochondrial surface in Drosophila and mammalian cells. Ribosomes stalled at the normal stop codon continue to add to the C-terminus of C-I30 certain amino acids non-coded by mRNA template. C-terminally-extended C-I30 is toxic when assembled into C-I and forms aggregates in the cytosol. Enhancing co-translational quality control prevents C-I30 C-terminal extension and rescues mitochondrial and neuromuscular degeneration in a Parkinson’s disease model. These findings emphasize the importance of efficient translation termination and reveal unexpected link between mitochondrial health and proteome homeostasis mediated by MISTERMINATE.

Rescuing stalled ribosomes A small percentage of bacterial mRNAs lack a stop codon. Ribosomes stall at the end of such mRNAs, and the buildup of stalled ribosomes can be lethal. The primary rescue mechanism, in which translation continues on a piece of RNA that contains a stop codon, is a drug target. However, bacteria have another backup plan. James et al. present structures that show that ArfA (alternative rescue factor A) substitutes for a stop codon by binding in the ribosomal mRNA channel and recruiting RF2 (release factor 2). It mediates conformational changes required for RF2 to catalyze peptide release. Science , this issue p. 1437

Wild plant resources represent a major source of important stress-resistance genes. We previously collected Xinjiang wild myrobalan plum (Prunus sogdiana) seeds from the wild natural forest in Daxigou, Huocheng County, Xinjiang, China and cultivated 139 individual P. sogdiana plants. After many years of scoring the resistance of these myrobalan plum plants to a root-knot nematode (RKN; Meloidogyne incognita), we identified stably resistant and susceptible plants. In this study, we used six resistant and six susceptible plants to identify molecular markers closely linked to the RKN resistance gene, Ma, and to investigate the characteristics of this gene in P. sogdiana. Of the two SCAR markers closely linked to the Ma gene, SCAL19690 was present in both resistant and susceptible plants, whereas SCAFLP2202 was not present in resistant or susceptible individuals, indicating that SCAL19690 and SCAFLP2202 could not be used as tools to screen for RKN resistance conferred by the Ma gene in P. sogdiana. We cloned the Ma gene and identified a stop codon at 523 bp in the mRNA of both resistant and susceptible individuals, which only encodes a 174-amino acid polypeptide in P. sogdiana due to premature translational termination, indicating that the Ma protein in P. sogdiana is incomplete. These results indicate that the Ma gene in Xinjiang wild myrobalan plum is different from that of European Myrobalan plum (P. cerasifera), demonstrating that wild myrobalan plum accessions of different origins represent rich, diverse genetic resources of resistance to M. incognita.

BACKGROUND Gastric cancer (GC) is the fifth most common cancer type worldwide. The aim of this study was to identify gastric-related therapeutic indicators on the basis of the ego network analysis. MATERIAL AND METHODS The microarray data related to GC was downloaded from ArrayExpress database. All human protein-protein interaction (PPI) networks were downloaded from the STRING database. Ego genes were identified on the basis of PPI networks and the gene expression in GC, and then co-expression networks (ego networks) were constructed using these ego genes. On the basis of ego networks, the optimal GO terms and genes were predicted by affinity predictions and cold read predictions. Finally, the predicted genes as effective biomarkers for GC were verified by the bioinformatics analysis. RESULTS The differential expression networks were conducted and comprised of 365 edges and 232 nodes, which resulted in 218 ego genes. Although there was no significant difference in the expression of top ten ego genes among different groups of GC samples, it was eventually confirmed that top three optimal GO terms with highest cool read values were translational termination (cool read value = 0.987), translational elongation (cool read value = 0.986), and macromolecular complex disassembly (cool read value = 0.985) and top five optimal genes were UBA52, RPS27A, MAPK1, UBC, and UBB. UBA52, RPS27A, and MAPK1 were verified by the bioinformatics analysis to be related to the progression and metastasis of GC. CONCLUSIONS An ego network analysis approach is a very effective method for screening GC and the screened genes might be biomarkers for GC diagnosis and treatment.

The maize (Zea mays) stigma, which is commonly known as silk, is indispensable for reproduction and thus for grain yield. Here, we isolated a spontaneous mutant sk-A7110, which completely lacks silk; scanning electron microscopy showed that the sk-A7110 pistils degenerated during late floret differentiation. Genetic analysis confirmed that this trait was controlled by a recessive nuclear gene and sk-A7110 was mapped to a 74.13-kb region on chromosome 2 between the simple sequence repeat markers LA714 and L277. Sequence analysis of candidate genes in this interval identified a single-nucleotide insertion at position 569 downstream of the transcriptional start site in Zm00001d002970, which encodes a UDP-glycosyltransferase; this insertion produces a frameshift and premature translational termination. RNA-sequencing analysis of young ears identified 258 differentially expressed genes (DEGs) between sk-A7110 and the wild type (WT), including 119 up- and 139 down-regulated genes. Interestingly, most DEGs related to jasmonic acid (JA) synthesis were up-regulated in the mutant compared to WT. Consistent with this, the JA and JA-Isoleucine (JA-Ile) contents were significantly higher in sk-A7110 ears than in WT. At the same time, RNA-sequencing analysis of tassels showed that sk-A7110 could reduce the number of tassel branches in maize by down-regulating the expression of UB2 and UB3 genes. Our identification of the sk-A7110 mutant and the responsible gene will facilitate further studies on female infertility research or maize breeding.

The universally conserved Gly-Gly-Gln (GGQ) tripeptide in release factors or release factor-like surveillance proteins is required to catalyze the release of nascent peptide in the ribosome. The glutamine of the GGQ is methylated post-translationally at the N5 position in vivo; this covalent modification is essential for optimal cell growth and efficient translation termination. However, the precise conformation of the methylated-GGQ tripeptide in the ribosome remains unknown. Using cryoEM and X-ray crystallography, we report the conformation of methylated-GGQ in the peptidyl transferase center of the ribosome during canonical translational termination and co-translation quality control. It has been suggested that the GGQ motif arose independently through convergent evolution among otherwise unrelated proteins that catalyze peptide release. The requirement for this tripeptide in the highly conserved peptidyl transferase center suggests that the conformation reported here is likely shared during termination of protein synthesis in all domains of life.

Background Nonsense mutations promote premature translational termination, introducing stop codons within the coding region of mRNAs and causing inherited diseases, including thalassemia. For instance, in β039 thalassemia the CAG (glutamine) codon is mutated to the UAG stop codon, leading to premature translation termination and to mRNA destabilization through the well described NMD (nonsense-mediated mRNA decay). In order to develop an approach facilitating translation and, therefore, protection from NMD, ribosomal read-through molecules, such as aminoglycoside antibiotics, have been tested on mRNAs carrying premature stop codons. These findings have introduced new hopes for the development of a pharmacological approach to the β039 thalassemia therapy. While several strategies, designed to enhance translational read-through, have been reported to inhibit NMD efficiency concomitantly, experimental tools for systematic analysis of mammalian NMD inhibition by translational read-through are lacking. Results We developed a human cellular model of the β039 thalassemia mutation with UPF-1 suppressed and showing a partial NMD suppression. Conclusions This novel cellular model could be used for the screening of molecules exhibiting preferential read-through activity allowing a great rescue of the mutated transcripts. Electronic supplementary material The online version of this article (10.1186/s12896-018-0435-0) contains supplementary material, which is available to authorized users.

2-oxogluatrate and Fe(II)-dependent oxygenase domain-containing protein 1 (OGFOD1) was recently revealed to be a proline hydroxylase of RPS23 for translational termination. However, OGFOD1 is nuclear, whereas translational termination occurs in the cytoplasm, raising the possibility of another function of OGFOD1 in the nucleus. In this study, we demonstrate that OGFOD1 is involved in cell cycle regulation. OGFOD1 knockdown in MDA-MB-231 breast cancer cells significantly impeded cell proliferation and resulted in the accumulation of G1 and G2/M cells by decreasing the mRNA levels of G1/S transition- and G2/M-related transcription factors and their target genes. We also confirmed that OGFOD1 is highly expressed in breast cancer tissues by bioinformatic analysis and immunohistochemistry. Thus, we propose that OGFOD1 is required for breast cancer cell proliferation and is associated with poor prognosis in breast cancer.

Steroid-resistant nephrotic syndrome (SRNS), a heterogeneous disorder of the renal glomerular filtration barrier, results in impairment of glomerular permselectivity. Inheritance of genetic SRNS may be autosomal dominant or recessive, with a subset of autosomal recessive SRNS presenting as congenital nephrotic syndrome (CNS). Mutations in 53 genes are associated with human SRNS, but these mutations explain ≤30% of patients with hereditary cases and only 20% of patients with sporadic cases. The proteins encoded by these genes are expressed in podocytes, and malfunction of these proteins leads to a universal end point of podocyte injury, glomerular filtration barrier disruption, and SRNS. Here, we identified novel disease-causing mutations in membrane-associated guanylate kinase, WW, and PDZ domain-containing 2 (MAGI2) through whole-exome sequencing of a deeply phenotyped cohort of patients with congenital, childhood-onset SRNS. Although MAGI2 has been shown to interact with nephrin and regulate podocyte cytoskeleton and slit diaphragm dynamics, MAGI2 mutations have not been described in human SRNS. We detected two unique frameshift mutations and one duplication in three patients (two families); two siblings shared the same homozygous frameshift mutation, whereas one individual with sporadic SRNS exhibited compound heterozygosity. Two mutations were predicted to introduce premature stop codons, and one was predicted to result in read through of the normal translational termination codon. Immunohistochemistry in kidney sections from these patients revealed that mutations resulted in lack of or diminished podocyte MAGI2 expression. Our data support the finding that mutations in the MAGI2 gene are causal for congenital SRNS.

Nonsense-mediated mRNA decay, or NMD, is a quality control mechanism that identifies cytoplasmic mRNAs containing translational termination (stop) codons in specific contexts-either premature termination codons or unusually long 3΄ untranslated regions (UTRs)-and targets them for degradation. In recent studies, researchers in different labs have knocked out important genes involved in NMD, the up-frameshift genes Upf2 and Upf3a, and one component of chromatoid bodies, the Tudor domain-containing protein Tdrd6, and examined the consequences for spermatogenesis. Disruption of Upf2 during early stages of spermatogenesis resulted in disappearance of nearly all spermatogenic cells through loss of NMD. However, disruption of Upf2 during postmeiotic stages resulted in decreased long 3΄ UTR-mediated NMD but no interruption of exon junction-associated NMD. This difference in NMD targeting is possibly due to increased expression of Upf3a in postmeiotic germ cells that antagonizes the functions of Upf3b and somehow favors long 3΄ UTR-mediated NMD. Tying these all together, loss of Tdrd6, a structural component of the germ cell-specific cytoplasmic structures called chromatoid bodies, also resulted in loss of long 3΄ UTR-mediated NMD by interfering with UPF1/UPF2 interactions, delocalizing UPF1, and destroying chromatoid body integrity. These results suggest that chromatoid bodies play a specialized role in modulating the NMD machinery in postmeiotic spermatids.

Summary Nonsense-mediated mRNA decay (NMD) is an essential quality control system that degrades aberrant transcripts containing premature termination codons and regulates the expression of several normal transcripts. Targets for NMD are selected during translational termination. If termination is slow, the UPF1 NMD factor binds the eRF3 protein of the termination complex and then recruits UPF2 and UPF3. Consequently, the UPF1-2-3 NMD complex induces SMG7-mediated degradation of the target mRNA. It is unknown how formation of the NMD complex and transcript degradation are linked in plants. Previously we have shown that the N- and C-terminal domains of UPF1 act redundantly and that the N-terminal domain is phosphorylated. To clarify the role of UPF1 phosphorylation in plant NMD, we generated UPF1 mutants and analyzed their phosphorylation status and the NMD competency of the mutants. We show that although several residues in the N-terminal domain of UPF1 are phosphorylated, only three phosphorylated amino acids, S3, S13 and T29, play a role in NMD. Moreover, we found that the C-terminal domain consists of redundant S/TQ-rich segments and that S1076 is involved in NMD. All NMD-relevant phosphorylation sites were in the S/TQ context. Co-localization and fluorescence resonance energy transfer–fluorescence lifetime imaging assays suggest that N-terminal and probably also C-terminal phosphorylated S/TQ residues are the binding platform for SMG7. Our data support the hypothesis that phosphorylation of UPF1 connects NMD complex formation and the SMG7-mediated target transcript degradation steps of NMD. SMG7 binds the phosphorylated S/TQ sites of the UPF1 component of the NMD complex, and then it induces the degradation of the NMD target.

Motility driven by rotational movement of flagella allows bacteria and archaea to seek favourable conditions and escape toxic ones. However, archaeal flagella share structural similarities with bacterial type IV pili rather than bacterial flagella. The Haloferax volcanii genome contains two flagellin genes, flgA1 and flgA2. While FlgA1 has been shown to be a major flagellin, the function of FlgA2 is elusive. In this study, it was determined that although FlgA2 by itself does not confer motility to non-motile ΔflgA1 Hfx. volcanii, a subset of these mutant cells contains a flagellum. Consistent with FlgA2 being assembled into functional flagella, FlgA1 expressed from a plasmid can only complement a ΔflgA1 strain when co-expressed with chromosomal or plasmid-encoded FlgA2. Surprisingly, a mutant strain lacking FlgA2, but expressing chromosomally encoded FlgA1, is hypermotile, a phenotype that is accompanied by an increased number of flagella per cell, as well as an increased flagellum length. Site-directed mutagenesis resulting in early translational termination of flgA2 suggests that the hypermotility of the ΔflgA2 strain is not due to transcriptional regulation. This, and the fact that plasmid-encoded FlgA2 expression in a ΔflgA2 strain does not reduce its hypermotility, suggests a possible regulatory role for FlgA2 that depends on the relative abundance of FlgA1. Taken together, our results indicate that FlgA2 plays both structural and regulatory roles in Hfx. volcanii flagella-dependent motility. Future studies will build upon the data presented here to elucidate the significance of the hypermotility of this ΔflgA2 mutant, and will illuminate the regulation and function of archaeal flagella.

Selenoprotein expression in Escherichia coli redefines specific single UGA codons from translational termination to selenocysteine (Sec) insertion. This process requires the presence of a Sec Insertion Sequence (SECIS) in the mRNA, which forms a secondary structure that binds a unique Sec-specific elongation factor that catalyzes Sec insertion at the predefined UGA instead of release factor 2-mediated termination. During overproduction of recombinant selenoproteins, this process nonetheless typically results in expression of UGA-truncated products together with the production of recombinant selenoproteins. Here, we found that premature termination can be fully avoided through a SECIS-dependent Sec-mediated suppression of UGG, thereby yielding either tryptophan or Sec insertion without detectable premature truncation. The yield of recombinant selenoprotein produced with this method approached that obtained with a classical UGA codon for Sec insertion. Sec-mediated suppression of UGG thus provides a novel method for selenoprotein production, as here demonstrated with rat thioredoxin reductase. The results also reveal that the E. coli selenoprotein synthesis machinery has the inherent capability to promote wobble decoding.

Background The three stop codons UAA, UAG, and UGA signal the termination of mRNA translation. As a result of a mechanism that is not adequately understood, they are normally used with unequal frequencies. Results In this work, we showed that selective forces and mutational biases drive stop codon usage in the human genome. We found that, in respect to sense codons, stop codon usage was affected by stronger selective forces but was less influenced by neutral mutational biases. UGA is the most frequent termination codon in human genome. However, UAA was the preferred stop codon in genes with high breadth of expression, high level of expression, AT-rich coding sequences, housekeeping functions, and in gene ontology categories with the largest deviation from expected stop codon usage. Selective forces associated with the breadth and the level of expression favoured AT-rich sequences in the mRNA region including the stop site and its proximal 3’-UTR, but acted with scarce effects on sense codons, generating two regions, upstream and downstream of the stop codon, with strongly different base composition. By favouring low levels of GC-content, selection promoted labile local secondary structures at the stop site and its proximal 3’-UTR. The compositional and structural context favoured by selection was surprisingly emphasized in the class of ribosomal proteins and was consistent with sequence elements that increase the efficiency of translational termination. Stop codons were also heterogeneously distributed among chromosomes by a mechanism that was strongly correlated with the GC-content of coding sequences. Conclusions In human genome, the nucleotide composition and the thermodynamic stability of stop codon site and its proximal 3’-UTR are correlated with the GC-content of coding sequences and with the breadth and the level of gene expression. In highly expressed genes stop codon usage is compositionally and structurally consistent with highly efficient translation termination signals. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-2692-4) contains supplementary material, which is available to authorized users.

Nonsense-mediated mRNA decay (NMD) is a widespread quality control mechanism in eukaryotic cells. It can recognize and degrade aberrant transcripts harbouring a premature translational termination codon (PTC), and thereby prevent the production of C-terminally truncated proteins which might be deleterious. Approximately, 30% of human genetic diseases are caused by transcripts containing PTCs. These transcripts are potential targets of NMD. As for monogenic diseases, NMD has effects on the phenotype or mode of inheritance. Here, we explain the mechanism of this surveillance pathway, and take several neuromuscular disorders as examples to discuss its influence for human monogenic diseases. The deeper understanding for NMD will shed light on the nosogenesis and therapies of monogenic diseases.

The tmRNA/SmpB system, which is almost universal in bacteria, rescues bacterial ribosomes stalled at the end of non-stop mRNAs (mRNAs lacking a stop codon). In addition, a few bacteria, including Escherichia coli, have developed a second two-component system as reported by Chadani et al. (2012). A small protein, ArfA of 55 amino acids (formerly called YdhL), mediates binding of release factor 2 to the ribosomal A site lacking a complete mRNA codon and thereby triggers translational termination and rescue of the stalled ribosome.

Sixteen Lactobacillus strains were screened using a plate agar containing glycodeoxycholic acid or taurodeoxycholic acid and quantitative BSH assay method, along with cholesterol-removing properties in vitro. Most BSH-positive strains tested were more efficient in hydrolyzing glycoconjugated bile salts than tauroconjugated bile salts. Among these strains, Lactobacillus plantarum isolate BBE7 had a relatively high BSH activity toward both bile salts and a higher cholesterol-removing activity (about 72.8 %) from cholesterol (100 μg/mL)-containing MRS culture, as compared with other tested strains. The bsh gene of this strain composed of about 43 kDa protein was cloned, sequenced, and overexpressed in Escherichia coli BL21 (DE3). The bsh gene contained a single ORF of 975 nucleotides flanked by a methionine start codon and translational termination codon and encoded a 324-amino-acid protein.

Congenital lipoid adrenal hyperplasia (lipoid CAH) is a rare autosomal recessive disorder of adrenal and gonadal steroidogenesis. It is most frequently caused by mutations in the steroidogenic acute regulatory protein (StAR) gene. Patients with lipoid CAH typically present with adrenal crisis in early infancy, and those with a 46,XY karyotype have female genitalia. However, it has been recently recognized that the phenotype can be quite variable, in that adrenal insufficiency is detected later in life and patients may have partially masculinized or even normal male genitalia. We report a patient assigned and reared as a female with a 46,XY karyotype and with a homozygous intron 2 (c.178+1G>C) splice site mutation of the StAR gene, which is a novel mutation that causes lipoid CAH. Her clinical presentation was somewhat atypical for a patient with classic lipoid CAH, marked by mild masculinization of the genitalia, detectable adrenal steroids at baseline, and ability to tolerate the stress of a surgical procedure with anesthesia without receiving glucocorticoid treatment. Conclusion: There is significant phenotypic variability among patients with lipoid CAH. While splice site mutations in the StAR gene lead to premature translational termination, resulting in truncated and non-functional proteins, there is phenotypic variability among patients with such mutations. Our patient appears to have the more atypical phenotype compared to reported patients with similar mutations. The molecular mechanism underlying this heterogeneity remains unclear.

SUMMARY Recent reports point to small soluble oligomers, rather than insoluble fibrils, of amyloid β (Aβ), as the primary toxic species in Alzheimer’s disease. Previously, we developed a low-throughput assay in yeast that is capable of detecting small Aβ42 oligomer formation. Specifically, Aβ42 fused to the functional release factor domain of yeast translational termination factor, Sup35p, formed sodium dodecyl sulfate (SDS)-stable low-n oligomers in living yeast, which impaired release factor activity. As a result, the assay for oligomer formation uses yeast growth to indicate restored release factor activity and presumably reduced oligomer formation. We now describe our translation of this assay into a high-throughput screen (HTS) for anti-oligomeric compounds. By doing so, we also identified two presumptive anti-oligomeric compounds from a sub-library of 12,800 drug-like small molecules. Subsequent biochemical analysis confirmed their anti-oligomeric activity, suggesting that this form of HTS is an efficient, sensitive and cost-effective approach to identify new inhibitors of Aβ42 oligomerization.

Stop codons have been exploited for genetic incorporation of unnatural amino acids (Uaas) in live cells, but their low incorporation efficiency, which is possibly due to competition from release factors, limits the power and scope of this technology. Here we show that the reportedly essential release factor 1 (RF1) can be knocked out from Escherichia coli by 'fixing' release factor 2 (RF2). The resultant strain JX33 is stable and independent, and it allows UAG to be reassigned from a stop signal to an amino acid when a UAG-decoding tRNA-synthetase pair is introduced. Uaas were efficiently incorporated at multiple UAG sites in the same gene without translational termination in JX33. We also found that amino acid incorporation at endogenous UAG codons is dependent on RF1 and mRNA context, which explains why E. coli tolerates apparent global suppression of UAG. JX33 affords a unique autonomous host for synthesizing and evolving new protein functions by enabling Uaa incorporation at multiple sites.

KEYWORDS Breast Cancer. Genetics. BRCA1. North Coastal Andhra Pradesh ABSTRACT Owing to its high incidence coupled with relatively good prognosis, breast cancer is the most prevalent cancer in the world today. Germ line mutations in the susceptibility gene BRCA1 in hereditary breast/ovarian cancer, though low in prevalence, are highly penetrant and show geographical variations. Most cancer-associated BRCA1 mutations identified to date result in the premature translational termination of the protein. However , the molecular and genetic effects of missense mutations remain largely unknown. There have been only a few reports from India on mutations in BRCA1 and none from North Coastal Andhra Pradesh. We have analyzed 114 breast cancer patients with (N = 9) and without (N = 105) a family history of breast cancer , 22 at risk relatives from familial (n=1 1), sporadic (n=11) cases and 97control subjects for mutations in exons 2 and 1 1 and their intron-exon boundaries of BRCA1 gene by direct sequencing. Sequence alignment was carried by CLUST AL W and PSI-BLAST. Out of eight sequence variants found, one novel deleterious frame-shift mutation (c.2717insA), one novel polymorphism (c.1400A>G), five previously reported common polymorphisms in exon 11 and one intronic (intron 1) variant (base1822C>T) were observed. All the identified polymorphisms in exon 11 fall in DNA binding domain of BRCA1 protein.

Yeast prion [PSI(+)] is a self-perpetuating amyloid of the translational termination factor Sup35. Although [PSI(+)] propagation is modulated by heat shock proteins (Hsps), high temperature was previously reported to have little or no effect on [PSI(+)]. Our results show that short-term exposure of exponentially growing yeast culture to mild heat shock, followed by immediate resumption of growth, leads to [PSI(+)] destabilization, sometimes persisting for several cell divisions after heat shock. Prion loss occurring in the first division after heat shock is preferentially detected in a daughter cell, indicating the impairment of prion segregation that results in asymmetric prion distribution between a mother cell and a bud. Longer heat shock or prolonged incubation in the absence of nutrients after heat shock led to [PSI(+)] recovery. Both prion destabilization and recovery during heat shock depend on protein synthesis. Maximal prion destabilization coincides with maximal imbalance between Hsp104 and other Hsps such as Hsp70-Ssa. Deletions of individual SSA genes increase prion destabilization and/or counteract recovery. The dynamics of prion aggregation during destabilization and recovery are consistent with the notion that efficient prion fragmentation and segregation require a proper balance between Hsp104 and other (e.g., Hsp70-Ssa) chaperones. In contrast to heat shock, [PSI(+)] destabilization by osmotic stressors does not always depend on cell proliferation and/or protein synthesis, indicating that different stresses may impact the prion via different mechanisms. Our data demonstrate that heat stress causes asymmetric prion distribution in a cell division and confirm that the effects of Hsps on prions are physiologically relevant.

Peppers (Capsicum spp.) display a variety of fruit colors that are reflected by the composition and amount of diverse carotenoid pigments accumulated in the pericarp. Three independent loci, c1, c2, and y, are known to determine the mature color of pepper fruits by their allelic combinations. We examined the inheritance of fruit color in recombinant inbred lines (RILs) derived from an interspecific cross between C. annuum cv. TF68 (red) and C. chinense cv. Habanero (orange). The c2 gene encodes phytoene synthase (PSY), a rate-limiting enzyme in the carotenoid biosynthesis pathway. TF68 has a dominant c2+ allele whereas Habanero is homozygous for the recessive c2 allele, which determined RIL fruit color. Here we report that the recessive c2 allele has a point mutation in the PSY gene that occurs at a splice acceptor site of the fifth intron leading to both a frame shift and premature translational termination, suggesting that impaired activity of PSY is responsible for orange fruit color. During ripening, PSY is expressed at a significantly high level in orange colored fruits compared to red ones. Interestingly, the PSY gene of red Habanero has a conserved splice acceptor dinucleotide AG. Further analysis suggests that red Habanero is a wild type revertant of the PSY mutant orange Habanero.

Viruses utilize a number of translational control mechanisms to regulate the relative expression levels of viral proteins on polycistronic mRNAs. One such mechanism, that of termination-dependent reinitiation, has been described in a number of both negative- and positive-strand RNA viruses. Dicistronic RNAs which exhibit termination–reinitiation typically have a start codon of the 3′-ORF (open reading frame) proximal to the stop codon of the upstream ORF. For example, the segment 7 RNA of influenza B is dicistronic, and the stop codon of the M1 ORF and the start codon of the BM2 ORF overlap in the pentanucleotide UAAUG (the stop codon of M1 is shown in bold and the start codon of BM2 is underlined). Recent evidence has highlighted the potential importance of mRNA–rRNA interactions in reinitiation on caliciviral and influenza B viral RNAs, probably used to tether 40S ribosomal subunits to the RNA after termination in time for initiation factors to be recruited to the AUG of the downstream ORF. The present review summarizes how such interactions regulate reinitiation in an array of RNA viruses, and discusses what is known about reinitiation in viruses that do not rely on apparent mRNA–rRNA interactions.

Protein synthesis (translation) stops at stop codons, codons not complemented by tRNA anticodons. tRNAs matching stops, antitermination (Ter) tRNAs, prevent translational termination, producing dysfunctional proteins. Genomes avoid tRNAs with anticodons whose complement (the anticodon of the 'antisense' tRNA) matches stops. This suggests that antisense tRNAs, which also form cloverleaves, are occasionally expressed. Mitochondrial antisense tRNA expression is plausible, because both DNA strands are transcribed as single RNAs, and tRNA structures signal RNA maturation. Results describe potential antisense Ter tRNAs in mammalian mitochondrial genomes detected by tRNAscan-SE, and evidence for adaptations preventing translational antitermination: genomes possessing Ter tRNAs use less corresponding stop codons; antisense Ter tRNAs form weaker cloverleaves than homologuous non-Ter antisense tRNAs; and genomic stop codon usages decrease with stabilities of codon-anticodon interactions and of Ter tRNA cloverleaves. This suggests that antisense tRNAs frequently function in translation. Results suggest that opposite strand coding is exceptional in modern genes, yet might be frequent for mitochondrial tRNAs. This adds antisense tRNA templating to other mitochondrial tRNA functions: sense tRNA templating, formation and regulation of secondary (light strand DNA) replication origins. Antitermination probably affects mitochondrial degenerative diseases and ageing: pathogenic mutations are twice as frequent in tRNAs with antisense Ter anticodons than in other tRNAs, and species lacking mitochondrial antisense Ter tRNAs have longer mean maximal lifespans than those possessing antisense Ter tRNAs.

In higher plants, male reproductive development is a complex biological process that includes cell division and differentiation, cell to cell communication etc., while the mechanism underlying plant male reproductive development remains less understood. GAMYB encodes a gibberellins acid (GA) inducible transcription factor that is required for the early anther development in rice (Oryza sativa L.). Here, we report the isolation and characterization of a new allele gamyb-4 with a C base deletion in the second exon (+2308), causing a frame shift and premature translational termination. Histological analysis showed that gamyb-4 developed abnormal enlarged tapetum and could not undergo normal meiosis. To understand the regulatory role of GAMYB, we carried out quantitative reverse transcription-polymerase chain reaction analysis and comparison of microarray data. These results revealed that the expression of TDR (TAPETUM DEGENERATION RETARDATION), a tapetal cell death regulator, was downregulated in gamyb-4 and udt1 (undeveloped tapetum1). While the GAMYB expression was not obviously changed in tdr and udt1-1, and no apparent expression fold change of UDT1 in tdr and gamyb-4, suggesting that TDR may act downstream of GAMYB and UDT1, and GAMYB and UDT1 work in parallel to regulate rice early anther development. This work is helpful in understanding the regulatory network in rice anther development.

We report the crystal structure of release factor 2 bound to ribosome with an aminoacyl tRNA substrate analog at the ribosomal P site, at 3.1 Å resolution. The structure shows that upon stop-codon recognition, the universally conserved GGQ motif packs tightly into the peptidyl transferase center. Nucleotide A2602 of 23S rRNA, implicated in peptide release, packs with the GGQ motif in release factor 2. The ribose of A76 of the peptidyl-tRNA adopts the C2′-endo conformation, and the 2′ hydroxyl of A76 is within hydrogen-bond distance of the 2′ hydroxyl of A2451. The structure suggests how a catalytic water can be coordinated in the peptidyl transferase center and, together with previous biochemical and computational data, suggests a model for how the ester bond between the peptidyl tRNA and the nascent peptide is hydrolyzed.