Membrane cholesterol modulates STEAP2 conformation during dynamic intracellular trafficking processes leading to broad subcellular distribution.

STEAP2 is a member of the Six-Transmembrane Epithelial Antigen of the Prostate (STEAP) protein family that is proposed to function as metalloreductase. While STEAP2 shows a complex subcellular distribution pattern localizing to both secretory and endocytic pathway organelles, how such broad steady-state distribution is maintained is unknown. Similarly, whether STEAP2 undergoes any compartment-specific modulation during intracellular trafficking has not been reported. Leveraging a newly-identified monoclonal antibody that recognizes a conformation-sensitive epitope nested in the second extracellular loop of STEAP2, we demonstrate that the epitope formation was dependent on the cholesterol content of the membrane in which STEAP2 was embedded. Monitoring the STEAP2-dependent internalization of this antibody uncovered STEAP2's rapid internalization from the cell surface and their subsequence trafficking to the Golgi region and endosome-like puncta. Acute inhibition of endocytosis also increased the detectable amount of STEAP2 at the plasma membrane. Collectively, these experiments demonstrate that an intricate balance of membrane flux between the secretory and endocytic pathways underlies the characteristic broad subcellular localization of STEAP2. By using a cell-based assay that detects the metalloreductase functions of cell surface-localizing STEAP4, STEAP2's metalloreductase activities were not detectable, suggesting that its enzymatic function is suppressed at the plasma membrane. The conformational modulation of STEAP2 by the local membrane cholesterol content can therefore serve as a potential mechanism to modulate STEAP2 function in a compartment-restricted manner, by coupling a pre-existing difference in cholesterol content among different cellular membranes to a dynamic trafficking process leading to broad subcellular distribution.

Effects of Pressure and pH on the Hydrolysis of Cytosine: Implications for Nucleotide Stability around Deep-Sea Black Smokers.

The relatively low chemical stability of cytosine compared with other nucleobases is a key concern in origin-of-life scenarios, but the effect of pressure on the rate of hydrolysis of cytosine to uracil remains unknown. Through in situ NMR spectroscopy measurements, it has been determined that the half-life of cytosine at 373.15 K decreases from (18.0±0.7) days at ambient pressure (0.1 MPa) to (8.64±0.18) days at high pressure (200 MPa). This yields an activation volume for hydrolysis of (-11.8±0.5) cm3 mol-1 ; a decrease that is similar to the molar volume of water (18.0 cm3 mol-1 ) and consistent with a tetrahedral 3,3-hydroxyamine transition-state/intermediate species. Similar behaviour was also observed for cytidine. At both ambient and high pressures, the half-life of cytosine decreases significantly as the pH decreases from 7.0 to 6.0. These results provide scant support for the notion that RNA-based life forms originated in high-temperature, high-pressure, acidic environments.

Improving phylogenetic inference of core Chlorophyta using chloroplast sequences with strong phylogenetic signals and heterogeneous models.

Phylogenetic relationships within the green algal phylum Chlorophyta have proven difficult to resolve. The core Chlorophyta include Chlorophyceae, Ulvophyceae, Trebouxiophyceae, Pedinophyceae and Chlorodendrophyceae, but the relationships among these classes remain unresolved and the monophyly of Ulvophyceae and Trebouxiophyceae are highly controversial. We analyzed a dataset of 101 green algal species and 73 protein-coding genes sampled from complete and partial chloroplast genomes, including six newly sequenced ulvophyte genomes (Blidingia minima NIES-1837, Ulothrix zonata, Halochlorococcum sp. NIES-1838, Scotinosphaera sp. NIES-154, Caulerpa brownii and Cephaleuros sp. HZ-2017). We applied the Tree Certainty (TC) score to quantify the level of incongruence between phylogenetic trees in chloroplast genomic datasets, and show that the conflicting phylogenetic trees of core Chlorophyta stem from the most GC-heterogeneous sites. With removing the most GC-heterogeneous sites, our chloroplast phylogenomic analyses using heterogeneous models consistently support monophyly of the Chlorophyceae and of the Trebouxiophyceae, but the Ulvophyceae was resolved as polyphyletic. Our analytical framework provides an efficient approach to reconstruct the optimal phylogenetic relationships by minimizing conflicting signals.

Does the Ribosome Challenge our Understanding of the RNA World?

In a recent article published in these pages, Bowman and colleagues propose that the ribosome represents a challenge to the RNA world model, a long-standing framework to explain the origin of DNA and genetically encoded proteins from a hypothetical RNA-based system. Specifically, they outline a scenario for the emergence and subsequent coevolution of the peptidyl transferase centre (PTC) of the ribosome with non-templated peptide products of this RNA through chemical evolution. They also propose that the PTC would have predated the emergence of enzymatic RNA replication, and that this in turn indicates that the RNA world never existed. We and others have previously incorporated non-templated peptide production as an early stage in the evolution of protein synthesis, which we would count as a chemical process, in agreement with Bowman and colleagues' model. However, their model raises an important question: to what extent could early protein synthesis and its products have evolved in the absence of Darwinian processes? We argue that evolution of the early ribosome requires Darwinian evolution, and that, while chemical evolution could give rise to peptidyl transferase activity, it is insufficient for subsequent improvement of a proto-PTC, or for ongoing coevolution of the proto-PTC with its early non-templated peptide products. We conclude that it is difficult to preclude the involvement of replicative processes, themselves subject to Darwinian evolution, from the evolution of the PTC. Finally, Bowman et al. call into question current models for the RNA to protein transition. We show that the difficulty that Bowman et al. have with this scenario is down to a misreading of our previous work.

Streptophyte algae and the origin of land plants revisited using heterogeneous models with three new algal chloroplast genomes.

The phylogenetic branching order of the green algal groups that gave rise to land plants remains uncertain despite its fundamental importance to understanding plant evolution. Previous studies have demonstrated that land plants evolved from streptophyte algae, but different lineages of streptophytes have been suggested to be the sister group of land plants. To better understand the evolutionary history of land plants and to determine the potential effects of "long-branch attraction" in phylogenetic reconstruction, we analyzed a chloroplast genome data set including three new chloroplast genomes from streptophyte algae: Coleochaetae orbicularis (Coleochaetales), Nitella hookeri (Charales), and Spirogyra communis (Zygnematales). We further applied a site pattern sorting method together with site- and time-heterogeneous models to investigate the branching order among streptophytes and land plants. Our chloroplast phylogenomic analyses support previous hypotheses based on nuclear data in placing Zygnematales alone, or a clade consisting of Coleochaetales plus Zygnematales, as the closest living relatives of land plants.

The relative ages of eukaryotes and akaryotes.

The Last Eukaryote Common Ancestor (LECA) appears to have the genetics required for meiosis, mitosis, nucleus and nuclear substructures, an exon/intron gene structure, spliceosomes, many centres of DNA replication, etc. (and including mitochondria). Most of these features are not generally explained by models for the origin of the Eukaryotic cell based on the fusion of an Archeon and a Bacterium. We find that the term 'prokaryote' is ambiguous and the non-phylogenetic term akaryote should be used in its place because we do not yet know the direction of evolution between eukaryotes and akaryotes. We use the term 'protoeukaryote' for the hypothetical stem group ancestral eukaryote that took up a bacterium as an endosymbiont that formed the mitochondrion. It is easier to make detailed models with a eukaryote to an akaryote transition, rather than vice versa. So we really are at a phylogenetic impasse in not being confident about the direction of change between eukaryotes and akaryotes.

Correcting the apparent mutation rate acceleration at shorter time scales under a Jukes-Cantor model.

At macroevolutionary time scales, and for a constant mutation rate, there is an expected linear relationship between time and the number of inferred neutral mutations (the "molecular clock"). However, at shorter time scales, a number of recent studies have observed an apparent acceleration in the rate of molecular evolution. We study this apparent acceleration under a Jukes-Cantor model applied to a randomly mating population, and show that, under the model, it arises as a consequence of ignoring short-term effects due to existing diversity within the population. The acceleration can be accounted for by adding the correction term h(0)e(-4µt/3) to the usual Jukes-Cantor formula p(t) = 3/4(1 - e (-(-4µt/3), where h(0) is the expected heterozygosity in the population at time t = 0. The true mutation rate µ may then be recovered, even if h(0) is not known, by estimating µ and h(0) simultaneously using least squares. Rate estimates made without the correction term (i.e., incorrectly assuming the population to be homogeneous) will result in a divergent rate curve of the form µ(div) = µ + C/t, so that the mutation rate appears to approach infinity as the time scale approaches zero. Although our quantitative results apply only to the Jukes-Cantor model, it is reasonable to suppose that the qualitative picture that emerges also applies to more complex models. Our study, therefore, demonstrates the importance of properly accounting for any ancestral diversity, because it may otherwise play a dominant role in rate overestimation.

Beyond phylogeny: pelecaniform and ciconiiform birds, and long-term niche stability.

Phylogenetic trees are a starting point for the study of further evolutionary and ecological questions. We show that for avian evolutionary relationships, improved taxon sampling, longer sequences and additional data sets are giving stability to the prediction of the grouping of pelecaniforms and ciconiiforms, thus allowing inferences to be made about long-term niche occupancy. Here we report the phylogeny of the pelecaniform birds and their water-carnivore allies using complete mitochondrial genomes, and show that the basic groupings agree with nuclear sequence phylogenies, even though many short branches are not yet fully resolved. In detail, we show that the Pelecaniformes (minus the tropicbird) and the Ciconiiformes (storks, herons and ibises) form a natural group within a seabird water-carnivore clade. We find pelicans are the closest relatives of the shoebill (in a clade with the hammerkop), and we confirm that tropicbirds are not pelecaniforms. In general, the group appears to be an adaptive radiation into an 'aquatic carnivore' niche that it has occupied for 60-70 million years. From an ecological and life history perspective, the combined pelecaniform-ciconiform group is more informative than focusing on differences in morphology. These findings allow a start to integrating molecular evolution and macroecology.

The RNA infrastructure: dark matter of the eukaryotic cell?

Eukaryotes express many functional non-protein-coding RNAs (ncRNAs) that participate in the processing and regulation of other RNA molecules. By focusing on connections between RNA-based processes, common patterns emerge that form a network-like RNA infrastructure. Owing to the intracellular movement of RNA during its processing (both between nuclear compartments and between the nucleus and cytoplasm), the RNA infrastructure contains both spatial and temporal connections. As research moves away from being protein-centric and focuses more on genomics, it is timely to explore these often 'hidden' aspects of the eukaryotic cell. The general and ancestral nature of most basic RNA-processing steps places a new focus on the generality of the spatial and temporal steps in RNA processing.

Characterization of RNase MRP RNA and novel snoRNAs from Giardia intestinalis and Trichomonas vaginalis.

BACKGROUND: Eukaryotic cells possess a complex network of RNA machineries which function in RNA-processing and cellular regulation which includes transcription, translation, silencing, editing and epigenetic control. Studies of model organisms have shown that many ncRNAs of the RNA-infrastructure are highly conserved, but little is known from non-model protists. In this study we have conducted a genome-scale survey of medium-length ncRNAs from the protozoan parasites Giardia intestinalis and Trichomonas vaginalis. RESULTS: We have identified the previously 'missing' Giardia RNase MRP RNA, which is a key ribozyme involved in pre-rRNA processing. We have also uncovered 18 new H/ACA box snoRNAs, expanding our knowledge of the H/ACA family of snoRNAs. CONCLUSIONS: Results indicate that Giardia intestinalis and Trichomonas vaginalis, like their distant multicellular relatives, contain a rich infrastructure of RNA-based processing. From here we can investigate the evolution of RNA processing networks in eukaryotes.

Origin of introns by 'intronization' of exonic sequences.

The mechanisms of spliceosomal intron creation have proved elusive. Here we describe a new mechanism: the recruitment of internal exonic sequences ('intronization') in Caenorhabditis species. The numbers of intronization events and introns gained by other mechanisms are similar, suggesting that intronization significantly contributes to recent intron creation in nematodes. Intronization is more common than the reverse process, loss of splicing of retained introns. Finally, these findings link alternative splicing with modern intron creation.

Tinamous and moa flock together: mitochondrial genome sequence analysis reveals independent losses of flight among ratites.

Ratites are large, flightless birds and include the ostrich, rheas, kiwi, emu, and cassowaries, along with extinct members, such as moa and elephant birds. Previous phylogenetic analyses of complete mitochondrial genome sequences have reinforced the traditional belief that ratites are monophyletic and tinamous are their sister group. However, in these studies ratite monophyly was enforced in the analyses that modeled rate heterogeneity among variable sites. Relaxing this topological constraint results in strong support for the tinamous (which fly) nesting within ratites. Furthermore, upon reducing base compositional bias and partitioning models of sequence evolution among protein codon positions and RNA structures, the tinamou-moa clade grouped with kiwi, emu, and cassowaries to the exclusion of the successively more divergent rheas and ostrich. These relationships are consistent with recent results from a large nuclear data set, whereas our strongly supported finding of a tinamou-moa grouping further resolves palaeognath phylogeny. We infer flight to have been lost among ratites multiple times in temporally close association with the Cretaceous-Tertiary extinction event. This circumvents requirements for transient microcontinents and island chains to explain discordance between ratite phylogeny and patterns of continental breakup. Ostriches may have dispersed to Africa from Eurasia, putting in question the status of ratites as an iconic Gondwanan relict taxon.

Phylogenetic tree reconstruction accuracy and model fit when proportions of variable sites change across the tree.

Commonly used phylogenetic models assume a homogeneous process through time in all parts of the tree. However, it is known that these models can be too simplistic as they do not account for nonhomogeneous lineage-specific properties. In particular, it is now widely recognized that as constraints on sequences evolve, the proportion and positions of variable sites can vary between lineages causing heterotachy. The extent to which this model misspecification affects tree reconstruction is still unknown. Here, we evaluate the effect of changes in the proportions and positions of variable sites on model fit and tree estimation. We consider 5 current models of nucleotide sequence evolution in a Bayesian Markov chain Monte Carlo framework as well as maximum parsimony (MP). We show that for a tree with 4 lineages where 2 nonsister taxa undergo a change in the proportion of variable sites tree reconstruction under the best-fitting model, which is chosen using a relative test, often results in the wrong tree. In this case, we found that an absolute test of model fit is a better predictor of tree estimation accuracy. We also found further evidence that MP is not immune to heterotachy. In addition, we show that increased sampling of taxa that have undergone a change in proportion and positions of variable sites is critical for accurate tree reconstruction.

Discovery of a potent and highly selective PDK1 inhibitor via fragment-based drug discovery.

We report the use of a fragment-based lead discovery method, Tethering with extenders, to discover a pyridinone fragment that binds in an adaptive site of the protein PDK1. With subsequent medicinal chemistry, this led to the discovery of a potent and highly selective inhibitor of PDK1, which binds in the 'DFG-out' conformation.

How old are RNA networks?

Some major classes of RNAs (such as mRNA, rRNA, tRNA and RNase P) are ubiquitous in all living systems so are inferred to have arisen early during the origin of life. However, the situation is not so clear for the system of RNA regulatory networks that continue to be uncovered, especially in eukaryotes. It is increasingly being recognised that networks of small RNAs are important for regulation in all cells, but it is not certain whether the origin of these networks are as old as rRNAs and tRNA. Another group of ncRNAs, including snoRNAs, occurs mainly in archaea and eukaryotes and their ultimate origin is less certain, although perhaps the simplest hypothesis is that they were present in earlier stages of life and were lost from bacteria. Some RNA networks may trace back to an early stage when there was just RNA and proteins, the RNP-world; before DNA.

Toward resolving deep neoaves phylogeny: data, signal enhancement, and priors.

We report three developments toward resolving the challenge of the apparent basal polytomy of neoavian birds. First, we describe improved conditional down-weighting techniques to reduce noise relative to signal for deeper divergences and find increased agreement between data sets. Second, we present formulae for calculating the probabilities of finding predefined groupings in the optimal tree. Finally, we report a significant increase in data: nine new mitochondrial (mt) genomes (the dollarbird, New Zealand kingfisher, great potoo, Australian owlet-nightjar, white-tailed trogon, barn owl, a roadrunner [a ground cuckoo], New Zealand long-tailed cuckoo, and the peach-faced lovebird) and together they provide data for each of the six main groups of Neoaves proposed by Cracraft J (2001). We use his six main groups of modern birds as priors for evaluation of results. These include passerines, cuckoos, parrots, and three other groups termed "WoodKing" (woodpeckers/rollers/kingfishers), "SCA" (owls/potoos/owlet-nightjars/hummingbirds/swifts), and "Conglomerati." In general, the support is highly significant with just two exceptions, the owls move from the "SCA" group to the raptors, particularly accipitrids (buzzards/eagles) and the osprey, and the shorebirds may be an independent group from the rest of the "Conglomerati". Molecular dating mt genomes support a major diversification of at least 12 neoavian lineages in the Late Cretaceous. Our results form a basis for further testing with both nuclear-coding sequences and rare genomic changes.

Coevolution of genomic intron number and splice sites.

Spliceosomal intron numbers and boundary sequences vary dramatically in eukaryotes. We found a striking correspondence between low intron number and strong sequence conservation of 5' splice sites (5'ss) across eukaryotic genomes. The phylogenetic pattern suggests that ancestral 5'ss were relatively weakly conserved, but that some lineages independently underwent both major intron loss and 5'ss strengthening. It seems that eukaryotic ancestors had relatively large intron numbers and 'weak' 5'ss, a pattern associated with frequent alternative splicing in modern organisms.

Two aspects along the continuum of pigeon evolution: A South-Pacific radiation and the relationship of pigeons within Neoaves.

Phylogenetics explores the continuum of shallower to deeper genetic divergences between taxa. Along this continuum increasing lengths of DNA sequence can be used to answer deeper and deeper questions about biological relationships. We use shorter, and then longer mitochondrial DNA sequences to address two aspects of pigeon evolution. Firstly, we examine the phylogenetic relationships of the eight genera within the South Pacific Ducula-Ptilinopus radiation, and examine how this radiation fits into pigeons generally. Within Ducula, taxa are closely related, whereas Ptilinopus is very diverse, and paraphyletic. One third of all pigeon species are within the Ducula-Ptilinopus radiation, however all are very similar ecologically. Secondly, we study the deeper phylogenetic question regarding the relationship of pigeons to other birds. To this end, we report the complete mitochondrial genome of Hemiphaganovaeseelandiae, a member of the Ducula-Ptilinopus radiation. We use this mitochondrial genome, along with additional sandgrouse (Pterocles namaqua) mitochondrial genes to assess various candidates for the closest relative of pigeons. Of parrots, shorebirds, and sandgrouse, we find highest support for the sandgrouse-pigeon grouping. Furthermore in these analyses the pigeon and sandgrouse group closer to the falcons than any other included taxon. The finding that pigeons and sandgrouse may be more closely related to falcons than to previous candidates such as shorebirds or parrots invites further investigation.