The tRNA aminoacylation co-factor Arc1p is excluded from the nucleus by an Xpo1p-dependent mechanism

Arc1p, a yeast tRNA-binding protein, forms a complex with the aminoacyl-tRNA synthetases, methionyl tRNA synthetase (MetRS) and glutamyl tRNA synthetase (GluRS). Although this complex localizes normally in the cytoplasm, in the absence of Arc1p the two free synthetases are also found inside the nucleus. In this work, in order to localize free Arc1 we abolished complex assembly by deleting the appended domains from both MetRS and GluRS. Surprisingly, free Arc1p remained cytoplasmic even when fitted with a strong nuclear localization signal (NLS). However, NLS-Arc1p accumulated in the nucleus when Xpo1/Crm1, the export receptor for NES-containing cargo proteins, was mutated. Thus, the cytoplasmic location of Arc1p is maintained by Xpo1p-dependent nuclear export and Arc1p could act as an adapter in the nucleocytoplasmic trafficking of tRNA and/or the tRNA-aminoacylation machinery.     

A pre-ribosome with a tadpole-like structure functions in ATP-dependent maturation of 60S subunits

Analyses of isolated pre-ribosomes yielded biochemical "snapshots" of the dynamic, nascent 60S and 40S subunits during their path from the nucleolus to the cytoplasm. Here, we present the structure of a pre-60S ribosomal intermediate located in the nucleoplasm. A huge dynein-related AAA-type ATPase (Rea1) and the Rix1 complex (Rix1-Ipi1-Ipi3) are components of an extended (approximately 45 nm long) pre-60S particle. Antibody crosslinking in combination with electron microscopy revealed that the Rea1 localizes to the "tail" region and ribosomal proteins to the "head" region of the elongated "tadpole-like" structure. Furthermore, in vitro treatment with ATP induces dissociation of Rea1 from the pre-60S subunits. Rea1 and the Rix1 complex could mediate ATP-dependent remodeling of 60S subunits and subsequent export from the nucleoplasm to the cytoplasm.     

Mutations in the YRB1 gene encoding yeast ran-binding-protein-1 that impair nucleocytoplasmic transport and suppress yeast mating defects

We identified two temperature-sensitive (ts) mutations in the essential gene, YRB1, which encodes the yeast homolog of Ran-binding-protein-1 (RanBP1), a known coregulator of the Ran GTPase cycle. Both mutations result in single amino acid substitutions of evolutionarily conserved residues (A91D and R127K, respectively) in the Ran-binding domain of Yrb1. The altered proteins have reduced affinity for Ran (Gsp1) in vivo. After shift to restrictive temperature, both mutants display impaired nuclear protein import and one also reduces poly(A)+ RNA export, suggesting a primary defect in nucleocytoplasmic trafficking. Consistent with this conclusion, both yrb1ts mutations display deleterious genetic interactions with mutations in many other genes involved in nucleocytoplasmic transport, including SRP1 (alpha-importin) and several beta-importin family members. These yrb1ts alleles were isolated by their ability to suppress two different types of mating-defective mutants (respectively, fus1Delta and ste5ts), indicating that reduction in nucleocytoplasmic transport enhances mating proficiency. Indeed, in both yrb1ts mutants, Ste5 (scaffold protein for the pheromone response MAPK cascade) is mislocalized to the cytosol, even in the absence of pheromone. Also, both yrb1ts mutations suppress the mating defect of a null mutation in MSN5, which encodes the receptor for pheromone-stimulated nuclear export of Ste5. Our results suggest that reimport of Ste5 into the nucleus is important in downregulating mating response.     

Evaluating the fitness of PA/I38T-substituted influenza A viruses with reduced baloxavir susceptibility in a competitive mixtures ferret model

Baloxavir is approved in several countries for the treatment of uncomplicated influenza in otherwise-healthy and high-risk patients. Treatment-emergent viruses with reduced susceptibility to baloxavir have been detected in clinical trials, but the likelihood of widespread occurrence depends on replication capacity and onward transmission. We evaluated the fitness of A/H3N2 and A/H1N1pdm09 viruses with the polymerase acidic (PA) I38T-variant conferring reduced susceptibility to baloxavir relative to wild-type (WT) viruses, using a competitive mixture ferret model, recombinant viruses and patient-derived virus isolates. The A/H3N2 PA/I38T virus showed a reduction in within-host fitness but comparable between-host fitness to the WT virus, while the A/H1N1pdm09 PA/I38T virus had broadly similar within-host fitness but substantially lower between-host fitness. Although PA/I38T viruses replicate and transmit between ferrets, our data suggest that viruses with this amino acid substitution have lower fitness relative to WT and this relative fitness cost was greater in A/H1N1pdm09 viruses than in A/H3N2 viruses.     

Estimating the Fitness Advantage Conferred by Permissive Neuraminidase Mutations in Recent Oseltamivir-Resistant A(H1N1)pdm09 Influenza Viruses

Oseltamivir is relied upon worldwide as the drug of choice for the treatment of human influenza infection. Surveillance for oseltamivir resistance is routinely performed to ensure the ongoing efficacy of oseltamivir against circulating viruses. Since the emergence of the pandemic 2009 A(H1N1) influenza virus (A(H1N1)pdm09), the proportion of A(H1N1)pdm09 viruses that are oseltamivir resistant (OR) has generally been low. However, a cluster of OR A(H1N1)pdm09 viruses, encoding the neuraminidase (NA) H275Y oseltamivir resistance mutation, was detected in Australia in 2011 amongst community patients that had not been treated with oseltamivir. Here we combine a competitive mixtures ferret model of influenza infection with a mathematical model to assess the fitness, both within and between hosts, of recent OR A(H1N1)pdm09 viruses. In conjunction with data from in vitro analyses of NA expression and activity we demonstrate that contemporary A(H1N1)pdm09 viruses are now more capable of acquiring H275Y without compromising their fitness, than earlier A(H1N1)pdm09 viruses circulating in 2009. Furthermore, using reverse engineered viruses we demonstrate that a pair of permissive secondary NA mutations, V241I and N369K, confers robust fitness on recent H275Y A(H1N1)pdm09 viruses, which correlated with enhanced surface expression and enzymatic activity of the A(H1N1)pdm09 NA protein. These permissive mutations first emerged in 2010 and are now present in almost all circulating A(H1N1)pdm09 viruses. Our findings suggest that recent A(H1N1)pdm09 viruses are now more permissive to the acquisition of H275Y than earlier A(H1N1)pdm09 viruses, increasing the risk that OR A(H1N1)pdm09 will emerge and spread worldwide.     

Avian biodiversity in the coastal wetlands of the Okahukura Peninsula

The Tapora Landcare Group, operating on the Okahukura Peninsula, has the long-term goal of making this region predator fenced. The aim of this study was to obtain information on the current status of avian biodiversity and the bird community across the band of coastal wetlands on the Okahukura Peninsula. Bird counts were conducted and playback lures used to detect three cryptic wetland species: fernbirds (Bowdleria punctata); spotless crakes (Porzana tabuensis); and banded rails (Gallirallus philippensis). Fernbirds and banded rails were detected at seven of the eight wetland sites sampled whereas spotless crakes were detected at two sites. The native species with the highest relative abundance across the eight sites were silvereyes (Zosterops lateralis) and South Island pied oystercatchers (Haematopus finschi). Changes in avian biodiversity over time in the region can now be monitored, and comprehensive long-term data on the status of avian biodiversity over time obtained.