A Molecular Portrait of De Novo Genes in Yeasts.

New genes, with novel protein functions, can evolve "from scratch" out of intergenic sequences. These de novo genes can integrate the cell's genetic network and drive important phenotypic innovations. Therefore, identifying de novo genes and understanding how the transition from noncoding to coding occurs are key problems in evolutionary biology. However, identifying de novo genes is a difficult task, hampered by the presence of remote homologs, fast evolving sequences and erroneously annotated protein coding genes. To overcome these limitations, we developed a procedure that handles the usual pitfalls in de novo gene identification and predicted the emergence of 703 de novo gene candidates in 15 yeast species from 2 genera whose phylogeny spans at least 100 million years of evolution. We validated 85 candidates by proteomic data, providing new translation evidence for 25 of them through mass spectrometry experiments. We also unambiguously identified the mutations that enabled the transition from noncoding to coding for 30 Saccharomyces de novo genes. We established that de novo gene origination is a widespread phenomenon in yeasts, only a few being ultimately maintained by selection. We also found that de novo genes preferentially emerge next to divergent promoters in GC-rich intergenic regions where the probability of finding a fortuitous and transcribed ORF is the highest. Finally, we found a more than 3-fold enrichment of de novo genes at recombination hot spots, which are GC-rich and nucleosome-free regions, suggesting that meiotic recombination contributes to de novo gene emergence in yeasts.

Genome-Scale Transcription-Translation Mapping Reveals Features of Zymomonas mobilis Transcription Units and Promoters.

Zymomonas mobilis is an ethanologenic alphaproteobacterium with promise for the industrial conversion of renewable plant biomass into fuels and chemical bioproducts. Limited functional annotation of the Z. mobilis genome is a current barrier to both fundamental studies of Z. mobilis and its development as a synthetic biology chassis. To gain insight, we collected sample-matched multiomics data, including RNA sequencing (RNA-seq), transcription start site (TSS) sequencing (TSS-seq), termination sequencing (term-seq), ribosome profiling, and label-free shotgun proteomic mass spectrometry, across different growth conditions and used these data to improve annotation and assign functional sites in the Z. mobilis genome. Proteomics and ribosome profiling informed revisions of protein-coding genes, which included 44 start codon changes and 42 added proteins. We developed statistical methods for annotating transcript 5' and 3' ends, enabling the identification of 3,940 TSSs and their corresponding promoters and 2,091 transcription termination sites, which were distinguished from RNA processing sites by the lack of an adjacent RNA 5' end. Our results revealed that Z. mobilis σA -35 and -10 promoter elements closely resemble canonical Escherichia coli -35 and -10 elements, with one notable exception: the Z. mobilis -10 element lacks the highly conserved -7 thymine observed in E. coli and other previously characterized σA promoters. The σA promoters of another alphaproteobacterium, Caulobacter crescentus, similarly lack the conservation of -7 thymine in their -10 elements. Our results anchor the development of Z. mobilis as a platform for synthetic biology and establish strategies for empirical genome annotation that can complement purely computational methods.IMPORTANCE Efforts to rationally engineer synthetic pathways in Zymomonas mobilis are impeded by a lack of knowledge and tools for predictable and quantitative programming of gene regulation at the transcriptional, posttranscriptional, and posttranslational levels. With the detailed functional characterization of the Z. mobilis genome presented in this work, we provide crucial knowledge for the development of synthetic genetic parts tailored to Z. mobilis This information is vital as researchers continue to develop Z. mobilis for synthetic biology applications. Our methods and statistical analyses also provide ways to rapidly advance the understanding of poorly characterized bacteria via empirical data that enable the experimental validation of sequence-based prediction for genome characterization and annotation.

Quantitative capillary zone electrophoresis-mass spectrometry reveals the N-glycome developmental plan during vertebrate embryogenesis.

Glycans are known to be involved in many biological processes, while little is known about the expression of N-glycans during vertebrate development. We now report the first quantitative studies of both the expression of N-linked glycans at six early development stages and the expression of N-glycosylated peptides at two early development stages in Xenopus laevis, the African clawed frog. N-Glycans were labeled with isobaric tandem mass tags, pooled, separated by capillary electrophoresis, and characterized using tandem mass spectrometry. We quantified 110 N-glycan compositions that spanned four orders of magnitude in abundance. Capillary electrophoresis was particularly useful in identifying charged glycans; over 40% of the observed glycan compositions were sialylated. The glycan expression was relatively constant until the gastrula-neurula transition (developmental stage 13), followed by massive reprogramming. An increase in oligomannosidic and a decrease in the paucimannosidic and phosphorylated oligomannosidic glycans were observed at the late tailbud stage (developmental stage 41). Two notable and opposing regulation events were detected for sialylated glycans. LacdiNAc and Lewis antigen features distinguished down-regulated sialylation from up-regulated species. The level of Lewis antigen decreased at later stages, which was validated by Aleuria aurantia lectin (AAL) and Ulex europaeus lectin (UEA-I) blots. We also used HPLC coupled with tandem mass spectrometry to identify 611 N-glycosylation sites on 350 N-glycoproteins at the early stage developmental stage 1 (fertilized egg), and 1682 N-glycosylation sites on 1023 N-glycoproteins at stage 41 (late tailbud stage). Over two thirds of the N-glycoproteins identified in the late tailbud stage are associated with neuron projection morphogenesis, suggesting a vital role of the N-glycome in neuronal development.

Multiplexed proteome analysis with neutron-encoded stable isotope labeling in cells and mice.

We describe a protocol for multiplexed proteomic analysis using neutron-encoded (NeuCode) stable isotope labeling of amino acids in cells (SILAC) or mice (SILAM). This method currently enables simultaneous comparison of up to nine treatment and control proteomes. Another important advantage over traditional SILAC/SILAM is that shorter labeling times are required. Exploiting the small mass differences that correspond to subtle differences in the neutron-binding energies of different isotopes, the amino acids used in NeuCode SILAC/SILAM differ in mass by just a few milliDaltons. Isotopologs of lysine are introduced into cells or mammals, via the culture medium or diet, respectively, to metabolically label the proteome. Labeling time is ∼2 weeks for cultured cells and 3-4 weeks for mammals. The proteins are then extracted, relevant samples are combined, and these are enzymatically digested with lysyl endopeptidase (Lys-C). The resultant peptides are chromatographically separated and then mass analyzed. During mass spectrometry (MS) data acquisition, high-resolution MS1 spectra (≥240,000 resolving power at m/z = 400) reveal the embedded isotopic signatures, enabling relative quantification, while tandem mass spectra, collected at lower resolutions, provide peptide identities. Both types of spectra are processed using NeuCode-enabled MaxQuant software. In total, the approximate completion time for the protocol is 3-5 weeks.

Author Correction: Phosphorylation Dynamics Dominate the Regulated Proteome during Early Xenopus Development.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Expression of novel "LOCGEF" isoforms of ARHGEF18 in eosinophils.

Genomic, transcriptomic and proteomic databases indicate that the N-terminal 322 residues encoded by the presumptive LOC100996504 gene, which is adjacent to the ARHGEF18 guanine nucleotide exchange factor gene on chromosome 19, constitute the N-terminal portion of a 1361-residue isoform of ARHGEF18, dubbed LOCGEF-X3. LOCGEF-X3 arises from the use of a leukocyte-specific alternative transcriptional start site and splicing that bypasses the initial noncoding exon of the canonical 1015-residue ARHGEF18 isoform, p114. Eosinophil LOCGEF-X3 was amplified and cloned, recombinant LOCGEF-X3 was expressed, and anti-ARHGEF18 antibody was found to recognize a band in immunoblots of eosinophil lysates that co-migrates with recombinant LOCGEF-X3. PCR of eosinophils revealed minor amounts of transcripts for X4 and X5 isoforms of LOCGEF that arise from differential splicing and differ from the X3 isoform at their extreme N-termini. No p114 transcript or protein band was detected in eosinophils. Immunostaining with anti-ARHGEF18 antibody revealed relocalization of LOCGEF and RHOA from the periphery of round unstimulated eosinophils to the 2 poles of eosinophils polarized by treatment with IL5, CCL11, or IL33 in suspension. Canonical p114 ARHGEF18 has been implicated in maintenance of epithelial cell polarity. We suggest that the "LOC" portion of LOCGEF, which is unlike any other protein domain, has unique functions in control of polarity in activated eosinophils and other leukocytes.

Conserved Lipid and Small-Molecule Modulation of COQ8 Reveals Regulation of the Ancient Kinase-like UbiB Family.

Human COQ8A (ADCK3) and Saccharomyces cerevisiae Coq8p (collectively COQ8) are UbiB family proteins essential for mitochondrial coenzyme Q (CoQ) biosynthesis. However, the biochemical activity of COQ8 and its direct role in CoQ production remain unclear, in part due to lack of known endogenous regulators of COQ8 function and of effective small molecules for probing its activity in vivo. Here, we demonstrate that COQ8 possesses evolutionarily conserved ATPase activity that is activated by binding to membranes containing cardiolipin and by phenolic compounds that resemble CoQ pathway intermediates. We further create an analog-sensitive version of Coq8p and reveal that acute chemical inhibition of its endogenous activity in yeast is sufficient to cause respiratory deficiency concomitant with CoQ depletion. Collectively, this work defines lipid and small-molecule modulators of an ancient family of atypical kinase-like proteins and establishes a chemical genetic system for further exploring the mechanistic role of COQ8 in CoQ biosynthesis.

Phosphorylation Dynamics Dominate the Regulated Proteome during Early Xenopus Development.

The earliest stages of animal development are largely controlled by changes in protein phosphorylation mediated by signaling pathways and cyclin-dependent kinases. In order to decipher these complex networks and to discover new aspects of regulation by this post-translational modification, we undertook an analysis of the X. laevis phosphoproteome at seven developmental stages beginning with stage VI oocytes and ending with two-cell embryos. Concurrent measurement of the proteome and phosphoproteome enabled measurement of phosphosite occupancy as a function of developmental stage. We observed little change in protein expression levels during this period. We detected the expected phosphorylation of MAP kinases, translational regulatory proteins, and subunits of APC/C that validate the accuracy of our measurements. We find that more than half the identified proteins possess multiple sites of phosphorylation that are often clustered, where kinases work together in a hierarchical manner to create stretches of phosphorylated residues, which may be a means to amplify signals or stabilize a particular protein conformation. Conversely, other proteins have opposing sites of phosphorylation that seemingly reflect distinct changes in activity during this developmental timeline.

A quantitative map of the liver mitochondrial phosphoproteome reveals posttranslational control of ketogenesis.

Mitochondria are dynamic organelles that play a central role in a diverse array of metabolic processes. Elucidating mitochondrial adaptations to changing metabolic demands and the pathogenic alterations that underlie metabolic disorders represent principal challenges in cell biology. Here, we performed multiplexed quantitative mass spectrometry-based proteomics to chart the remodeling of the mouse liver mitochondrial proteome and phosphoproteome during both acute and chronic physiological transformations in more than 50 mice. Our analyses reveal that reversible phosphorylation is widespread in mitochondria, and is a key mechanism for regulating ketogenesis during the onset of obesity and type 2 diabetes. Specifically, we have demonstrated that phosphorylation of a conserved serine on Hmgcs2 (S456) significantly enhances its catalytic activity in response to increased ketogenic demand. Collectively, our work describes the plasticity of this organelle at high resolution and provides a framework for investigating the roles of proteome restructuring and reversible phosphorylation in mitochondrial adaptation.