The Arabidopsis PeptideAtlas: Harnessing worldwide proteomics data to create a comprehensive community proteomics resource

We developed a resource, the Arabidopsis PeptideAtlas (www.peptideatlas.org/builds/arabidopsis/), to solve central questions about the Arabidopsis thaliana proteome, such as the significance of protein splice forms and post-translational modifications (PTMs), or simply to obtain reliable information about specific proteins. PeptideAtlas is based on published mass spectrometry (MS) data collected through ProteomeXchange and reanalyzed through a uniform processing and metadata annotation pipeline. All matched MS-derived peptide data are linked to spectral, technical, and biological metadata. Nearly 40 million out of ∼143 million MS/MS (tandem MS) spectra were matched to the reference genome Araport11, identifying ∼0.5 million unique peptides and 17,858 uniquely identified proteins (only isoform per gene) at the highest confidence level (false discovery rate 0.0004; 2 non-nested peptides ≥9 amino acid each), assigned canonical proteins, and 3,543 lower-confidence proteins. Physicochemical protein properties were evaluated for targeted identification of unobserved proteins. Additional proteins and isoforms currently not in Araport11 were identified that were generated from pseudogenes, alternative start, stops, and/or splice variants, and small Open Reading Frames; these features should be considered when updating the Arabidopsis genome. Phosphorylation can be inspected through a sophisticated PTM viewer. PeptideAtlas is integrated with community resources including TAIR, tracks in JBrowse, PPDB, and UniProtKB. Subsequent PeptideAtlas builds will incorporate millions more MS/MS data.

A web resource providing the global community with mass spectrometry-based Arabidopsis proteome information and its spectral, technical, and biological metadata integrated with TAIR and JBrowse.     

Phylogenetic Profiling: How Much Input Data Is Enough?

Phylogenetic profiling is a well-established approach for predicting gene function based on patterns of gene presence and absence across species. Much of the recent developments have focused on methodological improvements, but relatively little is known about the effect of input data size on the quality of predictions. In this work, we ask: how many genomes and functional annotations need to be considered for phylogenetic profiling to be effective? Phylogenetic profiling generally benefits from an increased amount of input data. However, by decomposing this improvement in predictive accuracy in terms of the contribution of additional genomes and of additional annotations, we observed diminishing returns in adding more than ∼100 genomes, whereas increasing the number of annotations remained strongly beneficial throughout. We also observed that maximising phylogenetic diversity within a clade of interest improves predictive accuracy, but the effect is small compared to changes in the number of genomes under comparison. Finally, we show that these findings are supported in light of the Open World Assumption, which posits that functional annotation databases are inherently incomplete. All the tools and data used in this work are available for reuse from http://lab.dessimoz.org/14_phylprof. Scripts used to analyse the data are available on request from the authors.     

SEED Servers: High-Performance Access to the SEED Genomes,Annotations, and Metabolic Models

The remarkable advance in sequencing technology and the rising interest in medical and environmental microbiology, biotechnology, and synthetic biology resulted in a deluge of published microbial genomes. Yet, genome annotation, comparison, and modeling remain a major bottleneck to the translation of sequence information into biological knowledge, hence computational analysis tools are continuously being developed for rapid genome annotation and interpretation. Among the earliest, most comprehensive resources for prokaryotic genome analysis, the SEED project, initiated in 2003 as an integration of genomic data and analysis tools, now contains >5,000 complete genomes, a constantly updated set of curated annotations embodied in a large and growing collection of encoded subsystems, a derived set of protein families, and hundreds of genome-scale metabolic models. Until recently, however, maintaining current copies of the SEED code and data at remote locations has been a pressing issue. To allow high-performance remote access to the SEED database, we developed the SEED Servers (http://www.theseed.org/servers): four network-based servers intended to expose the data in the underlying relational database, support basic annotation services, offer programmatic access to the capabilities of the RAST annotation server, and provide access to a growing collection of metabolic models that support flux balance analysis. The SEED servers offer open access to regularly updated data, the ability to annotate prokaryotic genomes, the ability to create metabolic reconstructions and detailed models of metabolism, and access to hundreds of existing metabolic models. This work offers and supports a framework upon which other groups can build independent research efforts. Large integrations of genomic data represent one of the major intellectual resources driving research in biology, and programmatic access to the SEED data will provide significant utility to a broad collection of potential users.     

AliSim: A Fast and Versatile Phylogenetic Sequence Simulator for the Genomic Era

Sequence simulators play an important role in phylogenetics. Simulated data has many applications, such as evaluating the performance of different methods, hypothesis testing with parametric bootstraps, and, more recently, generating data for training machine-learning applications. Many sequence simulation programmes exist, but the most feature-rich programmes tend to be rather slow, and the fastest programmes tend to be feature-poor. Here, we introduce AliSim, a new tool that can efficiently simulate biologically realistic alignments under a large range of complex evolutionary models. To achieve high performance across a wide range of simulation conditions, AliSim implements an adaptive approach that combines the commonly used rate matrix and probability matrix approaches. AliSim takes 1.4 h and 1.3 GB RAM to simulate alignments with one million sequences or sites, whereas popular software Seq-Gen, Dawg, and INDELible require 2–5 h and 50–500 GB of RAM. We provide AliSim as an extension of the IQ-TREE software version 2.2, freely available at www.iqtree.org, and a comprehensive user tutorial at http://www.iqtree.org/doc/AliSim.     

Phyletic Profiling with Cliques of Orthologs Is Enhanced by Signatures of Paralogy Relationships

New microbial genomes are sequenced at a high pace, allowing insight into the genetics of not only cultured microbes, but a wide range of metagenomic collections such as the human microbiome. To understand the deluge of genomic data we face, computational approaches for gene functional annotation are invaluable. We introduce a novel model for computational annotation that refines two established concepts: annotation based on homology and annotation based on phyletic profiling. The phyletic profiling-based model that includes both inferred orthologs and paralogs—homologs separated by a speciation and a duplication event, respectively—provides more annotations at the same average Precision than the model that includes only inferred orthologs. For experimental validation, we selected 38 poorly annotated Escherichia coli genes for which the model assigned one of three GO terms with high confidence: involvement in DNA repair, protein translation, or cell wall synthesis. Results of antibiotic stress survival assays on E. coli knockout mutants showed high agreement with our model''s estimates of accuracy: out of 38 predictions obtained at the reported Precision of 60%, we confirmed 25 predictions, indicating that our confidence estimates can be used to make informed decisions on experimental validation. Our work will contribute to making experimental validation of computational predictions more approachable, both in cost and time. Our predictions for 998 prokaryotic genomes include ∼400000 specific annotations with the estimated Precision of 90%, ∼19000 of which are highly specific—e.g. “penicillin binding,” “tRNA aminoacylation for protein translation,” or “pathogenesis”—and are freely available at http://gorbi.irb.hr/.     

A Novel CaM Kinase II Pathway Controls the Location of Neuropeptide Release from Caenorhabditis elegans Motor Neurons

Neurons release neuropeptides via the regulated exocytosis of dense core vesicles (DCVs) to evoke or modulate behaviors. We found that Caenorhabditis elegans motor neurons send most of their DCVs to axons, leaving very few in the cell somas. How neurons maintain this skewed distribution and the extent to which it can be altered to control DCV numbers in axons or to drive release from somas for different behavioral impacts is unknown. Using a forward genetic screen, we identified loss-of-function mutations in UNC-43 (CaM kinase II) that reduce axonal DCV levels by ∼90% and cell soma/dendrite DCV levels by ∼80%, leaving small synaptic vesicles largely unaffected. Blocking regulated secretion in unc-43 mutants restored near wild-type axonal levels of DCVs. Time-lapse video microscopy showed no role for CaM kinase II in the transport of DCVs from cell somas to axons. In vivo secretion assays revealed that much of the missing neuropeptide in unc-43 mutants is secreted via a regulated secretory pathway requiring UNC-31 (CAPS) and UNC-18 (nSec1). DCV cargo levels in unc-43 mutants are similarly low in cell somas and the axon initial segment, indicating that the secretion occurs prior to axonal transport. Genetic pathway analysis suggests that abnormal neuropeptide function contributes to the sluggish basal locomotion rate of unc-43 mutants. These results reveal a novel pathway controlling the location of DCV exocytosis and describe a major new function for CaM kinase II.     

Genome Destabilizing Mutator Alleles Drive Specific Mutational Trajectories in Saccharomyces cerevisiae

In addition to environmental factors and intrinsic variations in base substitution rates, specific genome-destabilizing mutations can shape the mutational trajectory of genomes. How specific alleles influence the nature and position of accumulated mutations in a genomic context is largely unknown. Understanding the impact of genome-destabilizing alleles is particularly relevant to cancer genomes where biased mutational signatures are identifiable. We first created a more complete picture of cellular pathways that impact mutation rate using a primary screen to identify essential Saccharomyces cerevisiae gene mutations that cause mutator phenotypes. Drawing primarily on new alleles identified in this resource, we measure the impact of diverse mutator alleles on mutation patterns directly by whole-genome sequencing of 68 mutation-accumulation strains derived from wild-type and 11 parental mutator genotypes. The accumulated mutations differ across mutator strains, displaying base-substitution biases, allele-specific mutation hotspots, and break-associated mutation clustering. For example, in mutants of POLα and the Cdc13–Stn1–Ten1 complex, we find a distinct subtelomeric bias for mutations that we show is independent of the target sequence. Together our data suggest that specific genome-instability mutations are sufficient to drive discrete mutational signatures, some of which share properties with mutation patterns seen in tumors. Thus, in a population of cells, genome-instability mutations could influence clonal evolution by establishing discrete mutational trajectories for genomes.     

A γ-Secretase Independent Role for Presenilin in Calcium Homeostasis Impacts Mitochondrial Function and Morphology in Caenorhabditis elegans

Mutations in the presenilin (PSEN) encoding genes (PSEN1 and PSEN2) occur in most early onset familial Alzheimer’s Disease. Despite the identification of the involvement of PSEN in Alzheimer’s Disease (AD) ∼20 years ago, the underlying role of PSEN in AD is not fully understood. To gain insight into the biological function of PSEN, we investigated the role of the PSEN homolog SEL-12 in Caenorhabditis elegans. Using genetic, cell biological, and pharmacological approaches, we demonstrate that mutations in sel-12 result in defects in calcium homeostasis, leading to mitochondrial dysfunction. Moreover, consistent with mammalian PSEN, we provide evidence that SEL-12 has a critical role in mediating endoplasmic reticulum (ER) calcium release. Furthermore, we found that in SEL-12-deficient animals, calcium transfer from the ER to the mitochondria leads to fragmentation of the mitochondria and mitochondrial dysfunction. Additionally, we show that the impact that SEL-12 has on mitochondrial function is independent of its role in Notch signaling, γ-secretase proteolytic activity, and amyloid plaques. Our results reveal a critical role for PSEN in mediating mitochondrial function by regulating calcium transfer from the ER to the mitochondria.     

Synapse-Assembly Proteins Maintain Synaptic Vesicle Cluster Stability and Regulate Synaptic Vesicle Transport in Caenorhabditis elegans

The functional integrity of neurons requires the bidirectional active transport of synaptic vesicles (SVs) in axons. The kinesin motor KIF1A transports SVs from somas to stable SV clusters at synapses, while dynein moves them in the opposite direction. However, it is unclear how SV transport is regulated and how SVs at clusters interact with motor proteins. We addressed these questions by isolating a rare temperature-sensitive allele of Caenorhabditis elegans unc-104 (KIF1A) that allowed us to manipulate SV levels in axons and dendrites. Growth at 20° and 14° resulted in locomotion rates that were ∼3 and 50% of wild type, respectively, with similar effects on axonal SV levels. Corresponding with the loss of SVs from axons, mutants grown at 14° and 20° showed a 10- and 24-fold dynein-dependent accumulation of SVs in their dendrites. Mutants grown at 14° and switched to 25° showed an abrupt irreversible 50% decrease in locomotion and a 50% loss of SVs from the synaptic region 12-hr post-shift, with no further decreases at later time points, suggesting that the remaining clustered SVs are stable and resistant to retrograde removal by dynein. The data further showed that the synapse-assembly proteins SYD-1, SYD-2, and SAD-1 protected SV clusters from degradation by motor proteins. In syd-1, syd-2, and sad-1 mutants, SVs accumulate in an UNC-104-dependent manner in the distal axon region that normally lacks SVs. In addition to their roles in SV cluster stability, all three proteins also regulate SV transport.     

Nucleoporin FG Domains Facilitate mRNP Remodeling at the Cytoplasmic Face of the Nuclear Pore Complex

Directional export of messenger RNA (mRNA) protein particles (mRNPs) through nuclear pore complexes (NPCs) requires multiple factors. In Saccharomyces cerevisiae, the NPC proteins Nup159 and Nup42 are asymmetrically localized to the cytoplasmic face and have distinct functional domains: a phenylalanine-glycine (FG) repeat domain that docks mRNP transport receptors and domains that bind the DEAD-box ATPase Dbp5 and its activating cofactor Gle1, respectively. We speculated that the Nup42 and Nup159 FG domains play a role in positioning mRNPs for the terminal mRNP-remodeling steps carried out by Dbp5. Here we find that deletion (Δ) of both the Nup42 and Nup159 FG domains results in a cold-sensitive poly(A)+ mRNA export defect. The nup42ΔFG nup159ΔFG mutant also has synthetic lethal genetic interactions with dbp5 and gle1 mutants. RNA cross-linking experiments further indicate that the nup42ΔFG nup159ΔFG mutant has a reduced capacity for mRNP remodeling during export. To further analyze the role of these FG domains, we replaced the Nup159 or Nup42 FG domains with FG domains from other Nups. These FG “swaps” demonstrate that only certain FG domains are functional at the NPC cytoplasmic face. Strikingly, fusing the Nup42 FG domain to the carboxy-terminus of Gle1 bypasses the need for the endogenous Nup42 FG domain, highlighting the importance of proximal positioning for these factors. We conclude that the Nup42 and Nup159 FG domains target the mRNP to Gle1 and Dbp5 for mRNP remodeling at the NPC. Moreover, these results provide key evidence that character and context play a direct role in FG domain function and mRNA export.     

Remarkably Divergent Regions Punctuate the Genome Assembly of the Caenorhabditis elegans Hawaiian Strain CB4856

The Hawaiian strain (CB4856) of Caenorhabditis elegans is one of the most divergent from the canonical laboratory strain N2 and has been widely used in developmental, population, and evolutionary studies. To enhance the utility of the strain, we have generated a draft sequence of the CB4856 genome, exploiting a variety of resources and strategies. When compared against the N2 reference, the CB4856 genome has 327,050 single nucleotide variants (SNVs) and 79,529 insertion–deletion events that result in a total of 3.3 Mb of N2 sequence missing from CB4856 and 1.4 Mb of sequence present in CB4856 but not present in N2. As previously reported, the density of SNVs varies along the chromosomes, with the arms of chromosomes showing greater average variation than the centers. In addition, we find 61 regions totaling 2.8 Mb, distributed across all six chromosomes, which have a greatly elevated SNV density, ranging from 2 to 16% SNVs. A survey of other wild isolates show that the two alternative haplotypes for each region are widely distributed, suggesting they have been maintained by balancing selection over long evolutionary times. These divergent regions contain an abundance of genes from large rapidly evolving families encoding F-box, MATH, BATH, seven-transmembrane G-coupled receptors, and nuclear hormone receptors, suggesting that they provide selective advantages in natural environments. The draft sequence makes available a comprehensive catalog of sequence differences between the CB4856 and N2 strains that will facilitate the molecular dissection of their phenotypic differences. Our work also emphasizes the importance of going beyond simple alignment of reads to a reference genome when assessing differences between genomes.     

The Principal Role of Ku in Telomere Length Maintenance Is Promotion of Est1 Association with Telomeres

Telomere length is tightly regulated in cells that express telomerase. The Saccharomyces cerevisiae Ku heterodimer, a DNA end-binding complex, positively regulates telomere length in a telomerase-dependent manner. Ku associates with the telomerase RNA subunit TLC1, and this association is required for TLC1 nuclear retention. Ku–TLC1 interaction also impacts the cell-cycle-regulated association of the telomerase catalytic subunit Est2 to telomeres. The promotion of TLC1 nuclear localization and Est2 recruitment have been proposed to be the principal role of Ku in telomere length maintenance, but neither model has been directly tested. Here we study the impact of forced recruitment of Est2 to telomeres on telomere length in the absence of Ku’s ability to bind TLC1 or DNA ends. We show that tethering Est2 to telomeres does not promote efficient telomere elongation in the absence of Ku–TLC1 interaction or DNA end binding. Moreover, restoration of TLC1 nuclear localization, even when combined with Est2 recruitment, does not bypass the role of Ku. In contrast, forced recruitment of Est1, which has roles in telomerase recruitment and activation, to telomeres promotes efficient and progressive telomere elongation in the absence of Ku–TLC1 interaction, Ku DNA end binding, or Ku altogether. Ku associates with Est1 and Est2 in a TLC1-dependent manner and enhances Est1 recruitment to telomeres independently of Est2. Together, our results unexpectedly demonstrate that the principal role of Ku in telomere length maintenance is to promote the association of Est1 with telomeres, which may in turn allow for efficient recruitment and activation of the telomerase holoenzyme.     

DNA Replication Checkpoint Signaling Depends on a Rad53–Dbf4 N-Terminal Interaction in Saccharomyces cerevisiae

Dbf4-dependent kinase (DDK) and cyclin-dependent kinase (CDK) are essential to initiate DNA replication at individual origins. During replication stress, the S-phase checkpoint inhibits the DDK- and CDK-dependent activation of late replication origins. Rad53 kinase is a central effector of the replication checkpoint and both binds to and phosphorylates Dbf4 to prevent late-origin firing. The molecular basis for the Rad53–Dbf4 physical interaction is not clear but occurs through the Dbf4 N terminus. Here we found that both Rad53 FHA1 and FHA2 domains, which specifically recognize phospho-threonine (pT), interacted with Dbf4 through an N-terminal sequence and an adjacent BRCT domain. Purified Rad53 FHA1 domain (but not FHA2) bound to a pT Dbf4 peptide in vitro, suggesting a possible phospho-threonine-dependent interaction between FHA1 and Dbf4. The Dbf4–Rad53 interaction is governed by multiple contacts that are separable from the Cdc5- and Msa1-binding sites in the Dbf4 N terminus. Importantly, abrogation of the Rad53–Dbf4 physical interaction blocked Dbf4 phosphorylation and allowed late-origin firing during replication checkpoint activation. This indicated that Rad53 must stably bind to Dbf4 to regulate its activity.     

The Mps1 Kinase Modulates the Recruitment and Activity of Cnn1CENP-T at Saccharomyces cerevisiae Kinetochores

Kinetochores are conserved protein complexes that bind the replicated chromosomes to the mitotic spindle and then direct their segregation. To better comprehend Saccharomyces cerevisiae kinetochore function, we dissected the phospho-regulated dynamic interaction between conserved kinetochore protein Cnn1^CENP-T, the centromere region, and the Ndc80 complex through the cell cycle. Cnn1 localizes to kinetochores at basal levels from G1 through metaphase but accumulates abruptly at anaphase onset. How Cnn1 is recruited and which activities regulate its dynamic localization are unclear. We show that Cnn1 harbors two kinetochore-localization activities: a C-terminal histone-fold domain (HFD) that associates with the centromere region and a N-terminal Spc24/Spc25 interaction sequence that mediates linkage to the microtubule-binding Ndc80 complex. We demonstrate that the established Ndc80 binding site in the N terminus of Cnn1, Cnn1^60–84, should be extended with flanking residues, Cnn1^25–91, to allow near maximal binding affinity to Ndc80. Cnn1 localization was proposed to depend on Mps1 kinase activity at Cnn1–S74, based on in vitro experiments demonstrating the Cnn1–Ndc80 complex interaction. We demonstrate that from G1 through metaphase, Cnn1 localizes via both its HFD and N-terminal Spc24/Spc25 interaction sequence, and deletion or mutation of either region results in anomalous Cnn1 kinetochore levels. At anaphase onset (when Mps1 activity decreases) Cnn1 becomes enriched mainly via the N-terminal Spc24/Spc25 interaction sequence. In sum, we provide the first in vivo evidence of Cnn1 preanaphase linkages with the kinetochore and enrichment of the linkages during anaphase.     

Resection Activity of the Sgs1 Helicase Alters the Affinity of DNA Ends for Homologous Recombination Proteins in Saccharomyces cerevisiae

The RecQ helicase family is critical during DNA damage repair, and mutations in these proteins are associated with Bloom, Werner, or Rothmund-Thompson syndromes in humans, leading to cancer predisposition and/or premature aging. In the budding yeast Saccharomyces cerevisiae, mutations in the RecQ homolog, SGS1, phenocopy many of the defects observed in the human syndromes. One challenge to studying RecQ helicases is that their disruption leads to a pleiotropic phenotype. Using yeast, we show that the separation-of-function allele of SGS1, sgs1-D664Δ, has impaired activity at DNA ends, resulting in a resection processivity defect. Compromising Sgs1 resection function in the absence of the Sae2 nuclease causes slow growth, which is alleviated by making the DNA ends accessible to Exo1 nuclease. Furthermore, fluorescent microscopy studies reveal that, when Sgs1 resection activity is compromised in sae2Δ cells, Mre11 repair foci persist. We suggest a model where the role of Sgs1 in end resection along with Sae2 is important for removing Mre11 from DNA ends during repair.     

Genetic Analysis of the Ribosome Biogenesis Factor Ltv1 of Saccharomyces cerevisiae

Ribosome biogenesis has been studied extensively in the yeast Saccharomyces cerevisiae. Yeast Ltv1 is a conserved 40S-associated biogenesis factor that has been proposed to function in small subunit nuclear export. Here we show that Ltv1 has a canonical leucine-rich nuclear export signal (NES) at its extreme C terminus that is both necessary for Crm1 interaction and Ltv1 export. The C terminus of Ltv1 can substitute for the NES in the 60S-export adapter Nmd3, demonstrating that it is a functional NES. Overexpression of an Ltv1 lacking its NES (Ltv1∆C13) was strongly dominant negative and resulted in the nuclear accumulation of RpS3-GFP; however, export of the pre-40S was not affected. In addition, expression of endogenous levels of Ltv1∆C protein complemented both the slow-growth phenotype and the 40S biogenesis defect of an ltv1 deletion mutant. Thus, if Ltv1 is a nuclear export adapter for the pre-40S subunit, its function must be fully redundant with additional export factors. The dominant negative phenotype of Ltv1∆NES overexpression was suppressed by co-overexpressing RpS3 and its chaperone, Yar1, or by deletion of the RpS3-binding site in Ltv1∆NES, suggesting that titration of RpS3 by Ltv1∆NES is deleterious in yeast. The dominant-negative phenotype did not correlate with a decrease in 40S levels but rather with a reduction in the polysome-to-monosome ratio, indicating reduced rates of translation. We suggest that titration of RpS3 by excess nuclear Ltv1 interferes with 40S function or with a nonribosomal function of RpS3.     

Genome sequence of the Lebeckia ambigua-nodulating “Burkholderia sprentiae” strain WSM5005T

“Burkholderia sprentiae” strain WSM5005^T is an aerobic, motile, Gram-negative, non-spore-forming rod that was isolated in Australia from an effective N₂-fixing root nodule of Lebeckia ambigua collected in Klawer, Western Cape of South Africa, in October 2007. Here we describe the features of “Burkholderia sprentiae” strain WSM5005^T, together with the genome sequence and its annotation. The 7,761,063 bp high-quality-draft genome is arranged in 8 scaffolds of 236 contigs, contains 7,147 protein-coding genes and 76 RNA-only encoding genes, and is one of 20 rhizobial genomes sequenced as part of the DOE Joint Genome Institute 2010 Community Sequencing Program.     

Somatic Point Mutation Calling in Low Cellularity Tumors

Somatic mutation calling from next-generation sequencing data remains a challenge due to the difficulties of distinguishing true somatic events from artifacts arising from PCR, sequencing errors or mis-mapping. Tumor cellularity or purity, sub-clonality and copy number changes also confound the identification of true somatic events against a background of germline variants. We have developed a heuristic strategy and software (http://www.qcmg.org/bioinformatics/qsnp/) for somatic mutation calling in samples with low tumor content and we show the superior sensitivity and precision of our approach using a previously sequenced cell line, a series of tumor/normal admixtures, and 3,253 putative somatic SNVs verified on an orthogonal platform.