PLoS Genetics

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ISSN / EISSN : 15537390 / 15537404
Current Publisher: Public Library of Science (PLoS) (10.1371)
Total articles ≅ 8,971
Google Scholar h5-index: 106
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Ujué Fresán, Maria A. Rodríguez-Sánchez, Oscar Reina, Victor G. Corces, M. Lluisa Espinàs
PLOS Genetics, Volume 16; doi:10.1371/journal.pgen.1008962

Abstract:
Haspin, a highly conserved kinase in eukaryotes, has been shown to be responsible for phosphorylation of histone H3 at threonine 3 (H3T3ph) during mitosis, in mammals and yeast. Here we report that haspin is the kinase that phosphorylates H3T3 in Drosophila melanogaster and it is involved in sister chromatid cohesion during mitosis. Our data reveal that haspin also phosphorylates H3T3 in interphase. H3T3ph localizes in broad silenced domains at heterochromatin and lamin-enriched euchromatic regions. Loss of haspin compromises insulator activity in enhancer-blocking assays and triggers a decrease in nuclear size that is accompanied by changes in nuclear envelope morphology. We show that haspin is a suppressor of position-effect variegation involved in heterochromatin organization. Our results also demonstrate that haspin is necessary for pairing-sensitive silencing and it is required for robust Polycomb-dependent homeotic gene silencing. Haspin associates with the cohesin complex in interphase, mediates Pds5 binding to chromatin and cooperates with Pds5-cohesin to modify Polycomb-dependent homeotic transformations. Therefore, this study uncovers an unanticipated role for haspin kinase in genome organization of interphase cells and demonstrates that haspin is required for homeotic gene regulation. Haspin is a highly conserved kinase in eukaryotes involved in chromosome organization during mitosis. In this study we demonstrated that haspin is also required to maintain proper chromatin organization during interphase. Our analyses showed that Drosophila haspin is necessary for insulator activity, nuclear architecture, heterochromatin organization and pairing-sensitive gene silencing. We further found that haspin modulates Pds5-cohesin association with chromatin and it is required for robust Polycomb-mediated homeotic gene silencing. Overall our findings reveal that haspin kinase is a key element in chromatin organization and thereby regulates gene expression.
Helen Strutt, David Strutt
PLoS Genetics, Volume 16; doi:10.1371/journal.pgen.1008820

Abstract:
The core planar polarity proteins are essential mediators of tissue morphogenesis, controlling both the polarised production of cellular structures and polarised tissue movements. During development the core proteins promote planar polarisation by becoming asymmetrically localised to opposite cell edges within epithelial tissues, forming intercellular protein complexes that coordinate polarity between adjacent cells. Here we describe a novel protein complex that regulates the asymmetric localisation of the core proteins in the Drosophila pupal wing. DAnkrd49 (an ankyrin repeat protein) and Bride of Doubletime (Bdbt, a non-canonical FK506 binding protein family member) physically interact, and regulate each other’s levels in vivo. Loss of either protein results in a reduction in core protein asymmetry and disruption of the placement of trichomes at the distal edge of pupal wing cells. Post-translational modifications are thought to be important for the regulation of core protein behaviour and their sorting to opposite cell edges. Consistent with this, we find that loss of DAnkrd49 or Bdbt leads to reduced phosphorylation of the core protein Dishevelled and to decreased Dishevelled levels both at cell junctions and in the cytoplasm. Bdbt has previously been shown to regulate activity of the kinase Discs Overgrown (Dco, also known as Doubletime or Casein Kinase Iε), and Dco itself has been implicated in regulating planar polarity by phosphorylating Dsh as well as the core protein Strabismus. We demonstrate that DAnkrd49 and Bdbt act as dominant suppressors of Dco activity. These findings support a model whereby Bdbt and DAnkrd49 act together to modulate the activity of Dco during planar polarity establishment. In many animal tissues, sheets of cells are polarised in the plane of the tissue, which is evident by the production of polarised structures, such as hairs on the fly wing that point in the same direction or cilia that beat in the same direction. One group of proteins controlling this coordinated polarity are the core planar polarity proteins, which localise asymmetrically within cells such that some core proteins localise to one cell end and others to the opposite cell end. It is thought that modifications such as phosphorylation may locally regulate core protein stability, and this promotes sorting of proteins to different cell ends. We identify two proteins, DAnkrd49 and Bdbt, that form a complex and regulate core protein asymmetry. Loss of either protein causes a reduction in overall levels of the core protein Dishevelled (Dsh), and a reduction in its phosphorylation. We provide evidence that the effect on core protein asymmetry is mediated via regulation of the kinase activity of Discs overgrown (Dco, also known as Doubletime/Casein Kinase Iε) by DAnkrd49 and Bdbt. We propose that modulation of Dco activity by DAnkrd49 and Bdbt is a key step in the sorting of core proteins to opposite cell ends.
Kiran Busayavalasa, Mario Ruiz, Ranjan Devkota, Marcus Ståhlman, Rakesh Bodhicharla, Emma Svensk, Nils-Olov Hermansson, Jan Borén, Marc Pilon
PLoS Genetics, Volume 16; doi:10.1371/journal.pgen.1008975

Abstract:
The C. elegans proteins PAQR-2 (a homolog of the human seven-transmembrane domain AdipoR1 and AdipoR2 proteins) and IGLR-2 (a homolog of the mammalian LRIG proteins characterized by a single transmembrane domain and the presence of immunoglobulin domains and leucine-rich repeats in their extracellular portion) form a complex that protects against plasma membrane rigidification by promoting the expression of fatty acid desaturases and the incorporation of polyunsaturated fatty acids into phospholipids, hence increasing membrane fluidity. In the present study, we leveraged a novel gain-of-function allele of PAQR-1, a PAQR-2 paralog, to carry out structure-function studies. We found that the transmembrane domains of PAQR-2 are responsible for its functional requirement for IGLR-2, that PAQR-1 does not require IGLR-2 but acts via the same pathway as PAQR-2, and that the divergent N-terminal cytoplasmic domains of the PAQR-1 and PAQR-2 proteins serve a regulatory function and may regulate access to the catalytic site of these proteins. We also show that overexpression of human AdipoR1 or AdipoR2 alone is sufficient to confer increased palmitic acid resistance in HEK293 cells, and thus act in a manner analogous to the PAQR-1 gain-of-function allele. Cells are enclosed within membranes primarily composed of fat. When membranes contain much saturated fats, they tend to become more rigid, as with butter. Conversely, when membranes are rich in unsaturated fats, they become more fluid, as with vegetable oils. Our goal is to better understand how cells monitor and adjust the composition and properties of their membranes. We focus on a small group of proteins found in all animals, and called AdipoR1 and AdipoR2 in humans, and PAQR-1 and PAQR-2 in the worm Caenorhabditis elegans. We now found a version of PAQR-1 that is more “active”, and promotes increased levels of unsaturated fats in membranes. By swapping different parts of the PAQR-1 protein with those of PAQR-2, we were able to determine which protein parts played which roles. We found that it is the transmembrane domains of PAQR-2 that dictate its requirements for another protein called IGLR-2 and that the intracellular domains of PAQR-1 and PAQR-2 play a regulatory role. These studies help understand how AdipoR1 and AdipoR2 regulate membrane composition in human cells, which is a vital function for us to thrive on diets that vary greatly in the types of fats that they contain.
Tiit Örd, Tarmo Puurand, Daima Örd, Tarmo Annilo, Märt Möls, Maido Remm, Tõnis Örd
PLoS Genetics, Volume 16; doi:10.1371/journal.pgen.1008981

Abstract:
Tribbles homolog 3 (TRIB3) is pseudokinase involved in intracellular regulatory processes and has been implicated in several diseases. In this article, we report that human TRIB3 promoter contains a 33-bp variable number tandem repeat (VNTR) and characterize the heterogeneity and function of this genetic element. Analysis of human populations around the world uncovered the existence of alleles ranging from 1 to 5 copies of the repeat, with 2-, 3- and 5-copy alleles being the most common but displaying considerable geographical differences in frequency. The repeated sequence overlaps a C/EBP-ATF transcriptional regulatory element and is highly conserved, but not repeated, in various mammalian species, including great apes. The repeat is however evident in Neanderthal and Denisovan genomes. Reporter plasmid experiments in human cell culture reveal that an increased copy number of the TRIB3 promoter 33-bp repeat results in increased transcriptional activity. In line with this, analysis of whole genome sequencing and RNA-Seq data from human cohorts demonstrates that the copy number of TRIB3 promoter 33-bp repeats is positively correlated with TRIB3 mRNA expression level in many tissues throughout the body. Moreover, the copy number of the TRIB3 33-bp repeat appears to be linked to known TRIB3 eQTL SNPs as well as TRIB3 SNPs reported in genetic association studies. Taken together, the results indicate that the promoter 33-bp VNTR constitutes a causal variant for TRIB3 expression variation between individuals and could underlie the results of SNP-based genetic studies. The current article is devoted to a previously undescribed genetic element residing in the promoter region of human TRIB3 gene and consisting of a variable number of tandemly repeated 33-bp segments. We found that human genome contains from 1 to 5 copies of the repeat and that the copy number distribution varies in human populations around the world. Importantly, an increased number of copies associated with increased expression of the TRIB3 gene in many tissues of the body, giving rise to variation in TRIB3 gene expression between individuals. Mechanistically, the 33-bp segment possesses the ability to activate transcription, and it becomes more potent as it is repeated. The 33-bp segment is repeated in Neanderthals and Denisovans, but not in great apes or other mammals, suggesting that the expansion of the 33-bp unit in the TRIB3 promoter occurred after the split of the hominin and chimpanzee lineages. The TRIB3 protein participates in multiple biological processes as a regulatory factor and is linked to several diseases. Thus, the importance of our study is to shed light on a new mechanism determining TRIB3 expression level variation between individuals, and, more generally, to highlight how tandem repeat variations can serve as regulatory variants.
Aurelio Reyes, Paola Favia, Sara Vidoni, Vittoria Petruzzella, Massimo Zeviani
PLOS Genetics, Volume 16; doi:10.1371/journal.pgen.1008923

Abstract:
Mitochondrial translation defects can be due to mutations affecting mitochondrial- or nuclear-encoded components. The number of known nuclear genes involved in mitochondrial translation has significantly increased in the past years. RCC1L (WBSCR16), a putative GDP/GTP exchange factor, has recently been described to interact with the mitochondrial large ribosomal subunit. In humans, three different RCC1L isoforms have been identified that originate from alternative splicing but share the same N-terminus, RCC1LV1, RCC1LV2 and RCC1LV3. All three isoforms were exclusively localized to mitochondria, interacted with its inner membrane and could associate with homopolymeric oligos to different extent. Mitochondrial immunoprecipitation experiments showed that RCC1LV1 and RCC1LV3 associated with the mitochondrial large and small ribosomal subunit, respectively, while no significant association was observed for RCC1LV2. Overexpression and silencing of RCC1LV1 or RCC1LV3 led to mitoribosome biogenesis defects that resulted in decreased translation. Indeed, significant changes in steady-state levels and distribution on isokinetic sucrose gradients were detected not only for mitoribosome proteins but also for GTPases, (GTPBP10, ERAL1 and C4orf14), and pseudouridylation proteins, (TRUB2, RPUSD3 and RPUSD4). All in all, our data suggest that RCC1L is essential for mitochondrial function and that the coordination of at least two isoforms is essential for proper ribosomal assembly. Mitochondria are essential organelles responsible for the production of most of the energy required by the eukaryotic cells and hence their proper function is essential for cell survival. About 1,500 proteins are estimated to be present in human mitochondria but new proteins are still being reported as mitochondrial. While most proteins present in mitochondria are synthesized in the cytosol and then imported into mitochondria, 13 proteins encoded in the mitochondrial DNA are synthesized inside mitochondria by a dedicated set of proteins. RCC1L (WBSCR16) has recently been described as a protein involved in mitochondrial protein synthesis and more precisely in mitochondrial ribosome biogenesis. Three different RCC1L transcripts resulting in three different isoforms have been described; however, their role in mitochondrial translation has not been fully studied. Here we show that all three isoforms are present in mitochondria and that at least two of them play a role in mitochondrial ribosome biogenesis though the interaction with specific proteins that play a role in the assembly of either the large or the small ribosome subunit. This protein is an example of how biological processes may be more complex than anticipated as different isoforms of the same gene can have different roles in the cell.
Vanessa Khemici, Julien Prados, Bianca Petrignani, Benjamin Di Nolfi, Elodie Bergé, Caroline Manzano, Caroline Giraud, Patrick Linder
PLoS Genetics, Volume 16; doi:10.1371/journal.pgen.1008779

Abstract:
Staphylococcus aureus is an opportunistic pathogen that can grow in a wide array of conditions: on abiotic surfaces, on the skin, in the nose, in planktonic or biofilm forms and can cause many type of infections. Consequently, S. aureus must be able to adapt rapidly to these changing growth conditions, an ability largely driven at the posttranscriptional level. RNA helicases of the DEAD-box family play an important part in this process. In particular, CshA, which is part of the degradosome, is required for the rapid turnover of certain mRNAs and its deletion results in cold-sensitivity. To understand the molecular basis of this phenotype, we conducted a large genetic screen isolating 82 independent suppressors of cold growth. Full genome sequencing revealed the fatty acid synthesis pathway affected in many suppressor strains. Consistent with that result, sublethal doses of triclosan, a FASII inhibitor, can partially restore growth of a cshA mutant in the cold. Overexpression of the genes involved in branched-chain fatty acid synthesis was also able to suppress the cold-sensitivity. Using gas chromatography analysis of fatty acids, we observed an imbalance of straight and branched-chain fatty acids in the cshA mutant, compared to the wild-type. This imbalance is compensated in the suppressor strains. Thus, we reveal for the first time that the cold sensitive growth phenotype of a DEAD-box mutant can be explained, at least partially, by an improper membrane composition. The defect correlates with an accumulation of the pyruvate dehydrogenase complex mRNA, which is inefficiently degraded in absence of CshA. We propose that the resulting accumulation of acetyl-CoA fuels straight-chained fatty acid production at the expense of the branched ones. Strikingly, addition of acetate into the medium mimics the cshA deletion phenotype, resulting in cold sensitivity suppressed by the mutations found in our genetic screen or by sublethal doses of triclosan. DEAD-box RNA helicases are highly conserved proteins found in all domains of life. By acting on RNA secondary structures they determine the fate of RNA from transcription to degradation. Bacterial DEAD-box RNA helicases are not essential under laboratory conditions but are required for fitness and under stress conditions. Whereas many DEAD-box protein mutants display a cold sensitive phenotype, the underlying mechanisms have been studied only in few cases and found to be associated with ribosome biogenesis. We aimed here to elucidate the cold sensitivity of a cshA mutant in the Gram-positive opportunist pathogen Staphylococcus aureus. Our study revealed for the first time that part of the cold sensitivity is related to the inability of the bacterium to adapt the cytoplasmic membrane to lower temperatures. We propose that straight-chain fatty acid synthesis, reduced to sustain growth at lower temperature, is maintained due to inefficient turn-over of the pyruvate dehydrogenase mRNA, leading to elevated acetyl-CoA levels. This study allowed us to unravel at least in part the cold sensitive phenotype and to show that the pyruvate dehydrogenase activity plays an important function in the regulation of fatty acid composition of the membrane, a process that remains poorly understood in Gram-positive bacteria.
Stefani Giacopazzi, Daniel Vong, Alice Devigne, Needhi Bhalla
PLOS Genetics, Volume 16; doi:10.1371/journal.pgen.1008904

Abstract:
The conserved ATPase, PCH-2/TRIP13, is required during both the spindle checkpoint and meiotic prophase. However, its specific role in regulating meiotic homolog pairing, synapsis and recombination has been enigmatic. Here, we report that this enzyme is required to proofread meiotic homolog interactions. We generated a mutant version of PCH-2 in C. elegans that binds ATP but cannot hydrolyze it: pch-2E253Q. In vitro, this mutant can bind a known substrate but is unable to remodel it. This mutation results in some non-homologous synapsis and impaired crossover assurance. Surprisingly, worms with a null mutation in PCH-2’s adapter protein, CMT-1, the ortholog of p31comet, localize PCH-2 to meiotic chromosomes, exhibit non-homologous synapsis and lose crossover assurance. The similarity in phenotypes between cmt-1 and pch-2E253Q mutants suggest that PCH-2 can bind its meiotic substrates in the absence of CMT-1, in contrast to its role during the spindle checkpoint, but requires its adapter to hydrolyze ATP and remodel them. The production of sperm and eggs for sexual reproduction depends on meiosis. During this specialized cell division, homologous chromosomes pair, synapse and undergo meiotic recombination so that they are linked by at least one chiasma to promote their proper segregation. How homologous chromosomes ensure that these important interactions are with the correct partner is currently unknown. Here, we show that PCH-2 and its adapter protein, CMT-1, proofread homolog interactions to promote their fidelity and proper meiotic chromosome segregation.
Alexandra Gruss
PLOS Genetics, Volume 16; doi:10.1371/journal.pgen.1008842

Thomas Baier, Nick Jacobebbinghaus, Alexander Einhaus, Kyle J. Lauersen, Olaf Kruse
PLoS Genetics, Volume 16; doi:10.1371/journal.pgen.1008944

Abstract:
Efficient nuclear transgene expression in the green microalga Chlamydomonas reinhardtii is generally hindered by low transcription rates. Introns can increase transcript abundance by a process called Intron-Mediated Enhancement (IME) in this alga and has been broadly observed in other eukaryotes. However, the mechanisms of IME in microalgae are poorly understood. Here, we identified 33 native introns from highly expressed genes in C. reinhardtii selected from transcriptome studies as well as 13 non-native introns. We investigated their IME capacities and probed the mechanism of action by modification of splice sites, internal sequence motifs, and position within transgenes. Several introns were found to elicit strong IME and found to be broadly applicable in different expression constructs. We determined that IME in C. reinhardtii exclusively occurs from introns within transcribed ORFs regardless of the promoter and is not induced by traditional enhancers of transcription. Our results elucidate some mechanistic details of IME in C. reinhardtii, which are similar to those observed in higher plants yet underly distinctly different induction processes. Our findings narrow the focus of targets responsible for algal IME and provides evidence that introns are underestimated regulators of C. reinhardtii nuclear gene expression. Although many genetic tools and basic transformation strategies exist for the model microalga Chlamydomonas reinhardtii, high-level genetic engineering with this organism is hindered by its inherent recalcitrance to foreign gene expression and limited knowledge of responsible expression regulators. In this work, we characterized the dynamics of 33 endogenous and 13 non-native introns and their effect on gene expression as artificial insertions into codon optimized transgenes. We found that introns from different origins have the capacity to increase gene expression rates. Intron-mediated enhancement was observed exclusively when these elements were placed in transcripts but not outside of ORFs. Insertion of different endogenous introns into coding sequences was found to positively affect expression rates through a synergy of additive transcription enhancement and exon length reduction, similar to those natively found in the C. reinhardtii genome. Our results indicate that intensive mRNA processing plays an underestimated role in the regulation of native gene expression in C. reinhardtii. In addition to internal sequence motifs, the localization of artificially introduced introns greatly affected transgene expression levels. This work is highly valuable to the greater microalgal and synthetic biology research communities and contributes to broadening our understanding of eukaryotic intron-mediated enhancement.
Danhua Jiang, Michael Borg, Zdravko J. Lorković, Sean A. Montgomery, Akihisa Osakabe, Ramesh Yelagandula, Elin Axelsson, Frédéric Berger
PLoS Genetics, Volume 16; doi:10.1371/journal.pgen.1008964

Abstract:
Chromatin regulation of eukaryotic genomes depends on the formation of nucleosome complexes between histone proteins and DNA. Histone variants, which are diversified by sequence or expression pattern, can profoundly alter chromatin properties. While variants in histone H2A and H3 families are well characterized, the extent of diversification of histone H2B proteins is less understood. Here, we report a systematic analysis of the histone H2B family in plants, which have undergone substantial divergence during the evolution of each major group in the plant kingdom. By characterising Arabidopsis H2Bs, we substantiate this diversification and reveal potential functional specialization that parallels the phylogenetic structure of emergent clades in eudicots. In addition, we identify a new class of highly divergent H2B variants, H2B.S, that specifically accumulate during chromatin compaction of dry seed embryos in multiple species of flowering plants. Our findings thus identify unsuspected diverse properties among histone H2B proteins in plants that has manifested into potentially novel groups of histone variants. In addition to well-studied variants from core histones families H2A and H3, we report that land plants diversified their H2B family, leading to specialized H2B variants with specific patterns of expression, genomic distributions and properties.
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