Molecular basis for the bifunctional Uba4-Urm1 sulfur-relay system in tRNA thiolation and ubiquitin-like conjugation.

The chemical modification of tRNA bases by sulfur is crucial to tune translation and to optimize protein synthesis. In eukaryotes, the ubiquitin-related modifier 1 (Urm1) pathway is responsible for the synthesis of 2-thiolated wobble uridine (U34 ). During the key step of the modification cascade, the E1-like activating enzyme ubiquitin-like protein activator 4 (Uba4) first adenylates and thiocarboxylates the C-terminus of its substrate Urm1. Subsequently, activated thiocarboxylated Urm1 (Urm1-COSH) can serve as a sulfur donor for specific tRNA thiolases or participate in ubiquitin-like conjugation reactions. Structural and mechanistic details of Uba4 and Urm1 have remained elusive but are key to understand the evolutionary branch point between ubiquitin-like proteins (UBL) and sulfur-relay systems. Here, we report the crystal structures of full-length Uba4 and its heterodimeric complex with its substrate Urm1. We show how the two domains of Uba4 orchestrate recognition, binding, and thiocarboxylation of the C-terminus of Urm1. Finally, we uncover how the catalytic domains of Uba4 communicate efficiently during the reaction cycle and identify a mechanism that enables Uba4 to protect itself against self-conjugation with its own product, namely activated Urm1-COSH

The exonuclease Xrn1 activates transcription and translation of mRNAs encoding membrane proteins

The highly conserved 5'-3' exonuclease Xrn1 regulates gene expression in eukaryotes by coupling nuclear DNA transcription to cytosolic mRNA decay. By integrating transcriptome-wide analyses of translation with biochemical and functional studies, we demonstrate an unanticipated regulatory role of Xrn1 in protein synthesis. Xrn1 promotes translation of a specific group of transcripts encoding membrane proteins. Xrnl-dependence for translation is linked to poor structural RNA contexts for translation initiation, is mediated by interactions with components of the translation initiation machinery and correlates with an Xrnl-dependence for mRNA localization at the endoplasmic reticulum, the translation compartment of membrane proteins. Importantly, for this group of mRNAs, Xrn1 stimulates transcription, mRNA translation and decay. Our results uncover a crosstalk between the three major stages of gene expression coordinated by Xrn1 to maintain appropriate levels of membrane proteins

Australian health care providers' views on opt-out HIV testing

Background: Opt-out HIV testing is a novel concept in Australia. In the opt-out approach, health care providers (HCPs) routinely test patients for HIV unless they explicitly decline or defer. Opt-out HIV testing is only performed with the patients' consent, but pre-test counselling is abbreviated. Australian national testing guidelines do not currently recommend opt-out HIV testing for the general population. Non-traditional approaches to HIV testing (such as opt-out) could identify HIV infections and facilitate earlier treatment, which is particularly important now that HIV is a chronic, manageable disease. Our aim was to explore HCPs' attitudes toward opt-out HIV testing in an Australian context, to further understanding of its acceptability and feasibility. Methods: In this qualitative study, we used purposeful sampling to recruit HCPs who were likely to have experience with HIV testing in Western Australia. We interviewed them using a semi-structured guide and used content analysis as per Graneheim to code the data. Codes were then merged into subcategories and finally themes that unified the underlying concepts. We refined these themes through discussion among the research team. Results: Twenty four HCPs participated. Eleven participants had a questioning attitude toward opt-out HIV testing, while eleven favoured the approach. The remaining two participants had more nuanced perspectives that incorporated some characteristics of the questioning and favouring attitudes. Participants' views about opt-out HIV testing largely fell into two contrasting themes: normalisation and routinisation versus exceptionalism; and a need for proof versus openness to new approaches. Conclusion: Most HCPs in this study had dichotomous attitudes toward opt-out HIV testing, reflecting contrasting analytical styles. While some HCPs viewed it favourably, with the perceived benefits outweighing the perceived costs, others preferred to have evidence of efficacy and cost-effectiveness

Combinations of newly confirmed Glioma-Associated loci link regions on chromosomes 1 and 9 to increased disease risk

<p>Abstract</p> <p>Background</p> <p>Glioblastoma multiforme (GBM) tends to occur between the ages of 45 and 70. This relatively early onset and its poor prognosis make the impact of GBM on public health far greater than would be suggested by its relatively low frequency. Tissue and blood samples have now been collected for a number of populations, and predisposing alleles have been sought by several different genome-wide association (GWA) studies. The Cancer Genome Atlas (TCGA) at NIH has also collected a considerable amount of data. Because of the low concordance between the results obtained using different populations, only 14 predisposing single nucleotide polymorphism (SNP) candidates in five genomic regions have been replicated in two or more studies. The purpose of this paper is to present an improved approach to biomarker identification.</p> <p>Methods</p> <p>Association analysis was performed with control of population stratifications using the EIGENSTRAT package, under the null hypothesis of "no association between GBM and control SNP genotypes," based on an additive inheritance model. Genes that are strongly correlated with identified SNPs were determined by linkage disequilibrium (LD) or expression quantitative trait locus (eQTL) analysis. A new approach that combines meta-analysis and pathway enrichment analysis identified additional genes.</p> <p>Results</p> <p>(i) A meta-analysis of SNP data from TCGA and the Adult Glioma Study identifies 12 predisposing SNP candidates, seven of which are reported for the first time. These SNPs fall in five genomic regions (5p15.33, 9p21.3, 1p21.2, 3q26.2 and 7p15.3), three of which have not been previously reported. (ii) 25 genes are strongly correlated with these 12 SNPs, eight of which are known to be cancer-associated. (iii) The relative risk for GBM is highest for risk allele combinations on chromosomes 1 and 9. (iv) A combined meta-analysis/pathway analysis identified an additional four genes. All of these have been identified as cancer-related, but have not been previously associated with glioma. (v) Some SNPs that do not occur reproducibly across populations are in reproducible (invariant) pathways, suggesting that they affect the same biological process, and that population discordance can be partially resolved by evaluating processes rather than genes.</p> <p>Conclusion</p> <p>We have uncovered 29 glioma-associated gene candidates; 12 of them known to be cancer related (<it>p </it>= 1. 4 × 10^-6), providing additional statistical support for the relevance of the new candidates. This additional information on risk loci is potentially important for identifying Caucasian individuals at risk for glioma, and for assessing relative risk.</p

Outcome analysis following removal of locking plate fixation of the proximal humerus

<p>Abstract</p> <p>Background</p> <p>Concerning surgical management experience with locking plates for proximal humeral fractures has been described with promising results. Though, distinct hardware related complaints after fracture union are reported. Information concerning the outcome after removal of hardware from the proximal humerus is lacking and most studies on hardware removal are focused on the lower extremity. Therefore the aim of this study was to analyze the functional short-term outcome following removal of locking plate fixation of the proximal humerus.</p> <p>Methods</p> <p>Patients undergoing removal of a locking plate of the proximal humerus were prospectively followed. Patients were subdivided into the following groups: Group HI: symptoms of hardware related subacromial impingement, Group RD: persisting rotation deficit, Group RQ: patients with request for a hardware removal. The clinical (Constant-Murley score) and radiologic (AP and axial view) follow-up took place three and six months after the operation. To evaluate subjective results, the Medical Outcomes Study Short Form-36 (SF-36), was completed.</p> <p>Results</p> <p>59 patients were included. The mean length of time with the hardware in place was 15.2 ± 3.81 months. The mean of the adjusted overall Constant score before hardware removal was 66.2 ± 25.2% and increased significantly to 73.1 ± 22.5% after 3 months; and to 84.3 ± 20.6% after 6 months (p < 0.001). The mean of preoperative pain on the VAS-scale before hardware removal was 5.2 ± 2.9, after 6 months pain in all groups decreased significantly (p < 0.001). The SF-36 physical component score revealed a significant overall improvement in both genders (p < 0.001) at six months.</p> <p>Conclusion</p> <p>A significant improvement of clinical outcome following removal was found. However, a general recommendation for hardware removal is not justified, as the risk of an anew surgical and anesthetic procedure with all possible complications has to be carefully taken into account. However, for patients with distinct symptoms it might be justified.</p

Multiple TORC1-Associated Proteins Regulate Nitrogen Starvation-Dependent Cellular Differentiation in Saccharomyces cerevisiae

The budding yeast Saccharomyces cerevisiae undergoes differentiation into filamentous-like forms and invades the growth medium as a foraging response to nutrient and environmental stresses. These developmental responses are under the downstream control of effectors regulated by the cAMP/PKA and MAPK pathways. However, the upstream sensors and signals that induce filamentous growth through these signaling pathways are not fully understood. Herein, through a biochemical purification of the yeast TORC1 (Target of Rapamycin Complex 1), we identify several proteins implicated in yeast filamentous growth that directly associate with the TORC1 and investigate their roles in nitrogen starvation-dependent or independent differentiation in yeast.We isolated the endogenous TORC1 by purifying tagged, endogenous Kog1p, and identified associated proteins by mass spectrometry. We established invasive and pseudohyphal growth conditions in two S. cerevisiae genetic backgrounds (Σ1278b and CEN.PK). Using wild type and mutant strains from these genetic backgrounds, we investigated the roles of TORC1 and associated proteins in nitrogen starvation-dependent diploid pseudohyphal growth as well as nitrogen starvation-independent haploid invasive growth.We show that several proteins identified as associated with the TORC1 are important for nitrogen starvation-dependent diploid pseudohyphal growth. In contrast, invasive growth due to other nutritional stresses was generally not affected in mutant strains of these TORC1-associated proteins. Our studies suggest a role for TORC1 in yeast differentiation upon nitrogen starvation. Our studies also suggest the CEN.PK strain background of S. cerevisiae may be particularly useful for investigations of nitrogen starvation-induced diploid pseudohyphal growth

A mutated dph3 gene causes sensitivity of Schizosaccharomyces pombe cells to cytotoxic agents

Dph3 is involved in diphthamide modification of the eukaryotic translation elongation factor eEF2 and in Elongator-mediated modifications of tRNAs, where a 5-methoxycarbonyl-methyl moiety is added to wobble uridines. Lack of such modifications affects protein synthesis due to inaccurate translation of mRNAs at ribosomes. We have discovered that integration of markers at the msh3 locus of Schizosaccharomyces pombe impaired the function of the nearby located dph3 gene. Such integrations rendered cells sensitive to the cytotoxic drugs hydroxyurea and methyl methanesulfonate. We constructed dph3 and msh3 strains with mutated ATG start codons (ATGmut), which allowed investigating drug sensitivity without potential interference by marker insertions. The dph3- ATGmut and a dph3::loxP-ura4-loxM gene disruption strain, but not msh3-ATGmut, turned out to be sensitive to hydroxyurea and methyl methanesulfonate, likewise the strains with cassettes integrated at the msh3 locus. The fungicide sordarin, which inhibits diphthamide modified eEF2 of Saccharomyces cerevisiae, barely affected survival of wild type and msh3Δ S. pombe cells, while the dph3Δ mutant was sensitive. The msh3-ATG mutation, but not dph3Δ or the dph3-ATG mutation caused a defect in mating-type switching, indicating that the ura4 marker at the dph3 locus did not interfere with Msh3 function. We conclude that Dph3 is required for cellular resistance to the fungicide sordarin and to the cytotoxic drugs hydroxyurea and methyl methanesulfonate. This is likely mediated by efficient translation of proteins in response to DNA damage and replication stress

SAS6-like protein in Plasmodium indicates that conoid-associated apical complex proteins persist in invasive stages within the mosquito vector

The SAS6-like (SAS6L) protein, a truncated paralogue of the ubiquitous basal body/centriole protein SAS6, has been characterised recently as a flagellum protein in trypanosomatids, but associated with the conoid in apicomplexan Toxoplasma. The conoid has been suggested to derive from flagella parts, but is thought to have been lost from some apicomplexans including the malaria-causing genus Plasmodium. Presence of SAS6L in Plasmodium, therefore, suggested a possible role in flagella assembly in male gametes, the only flagellated stage. Here, we have studied the expression and role of SAS6L throughout the Plasmodium life cycle using the rodent malaria model P. berghei. Contrary to a hypothesised role in flagella, SAS6L was absent during gamete flagellum formation. Instead, SAS6L was restricted to the apical complex in ookinetes and sporozoites, the extracellular invasive stages that develop within the mosquito vector. In these stages SAS6L forms an apical ring, as we show is also the case in Toxoplasma tachyzoites. The SAS6L ring was not apparent in blood-stage invasive merozoites, indicating that the apical complex is differentiated between the different invasive forms. Overall this study indicates that a conoid-associated apical complex protein and ring structure is persistent in Plasmodium in a stage-specific manner

Defects in tRNA Modification Associated with Neurological and Developmental Dysfunctions in Caenorhabditis elegans Elongator Mutants

Elongator is a six subunit protein complex, conserved from yeast to humans. Mutations in the human Elongator homologue, hELP1, are associated with the neurological disease familial dysautonomia. However, how Elongator functions in metazoans, and how the human mutations affect neural functions is incompletely understood. Here we show that in Caenorhabditis elegans, ELPC-1 and ELPC-3, components of the Elongator complex, are required for the formation of the 5-carbamoylmethyl and 5-methylcarboxymethyl side chains of wobble uridines in tRNA. The lack of these modifications leads to defects in translation in C. elegans. ELPC-1::GFP and ELPC-3::GFP reporters are strongly expressed in a subset of chemosensory neurons required for salt chemotaxis learning. elpc-1 or elpc-3 gene inactivation causes a defect in this process, associated with a posttranscriptional reduction of neuropeptide and a decreased accumulation of acetylcholine in the synaptic cleft. elpc-1 and elpc-3 mutations are synthetic lethal together with those in tuc-1, which is required for thiolation of tRNAs having the 5′methylcarboxymethyl side chain. elpc-1; tuc-1 and elpc-3; tuc-1 double mutants display developmental defects. Our results suggest that, by its effect on tRNA modification, Elongator promotes both neural function and development