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An atlas of alternative splicing profiles and functional associations reveals new regulatory programs and genes that simultaneously express multiple major isoforms.

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An atlas of alternative splicing profiles and functional associations reveals new regulatory programs and genes that simultaneously express multiple major isoforms.

Genome Res. 2017 Oct;27(10):1759-1768

Authors: Tapial J, Ha KCH, Sterne-Weiler T, Gohr A, Braunschweig U, Hermoso-Pulido A, Quesnel-Vallières M, Permanyer J, Sodaei R, Marquez Y, Cozzuto L, Wang X, Gómez-Velázquez M, Rayon T, Manzanares M, Ponomarenko J, Blencowe BJ, Irimia M

Abstract
Alternative splicing (AS) generates remarkable regulatory and proteomic complexity in metazoans. However, the functions of most AS events are not known, and programs of regulated splicing remain to be identified. To address these challenges, we describe the Vertebrate Alternative Splicing and Transcription Database (VastDB), the largest resource of genome-wide, quantitative profiles of AS events assembled to date. VastDB provides readily accessible quantitative information on the inclusion levels and functional associations of AS events detected in RNA-seq data from diverse vertebrate cell and tissue types, as well as developmental stages. The VastDB profiles reveal extensive new intergenic and intragenic regulatory relationships among different classes of AS and previously unknown and conserved landscapes of tissue-regulated exons. Contrary to recent reports concluding that nearly all human genes express a single major isoform, VastDB provides evidence that at least 48% of multiexonic protein-coding genes express multiple splice variants that are highly regulated in a cell/tissue-specific manner, and that >18% of genes simultaneously express multiple major isoforms across diverse cell and tissue types. Isoforms encoded by the latter set of genes are generally coexpressed in the same cells and are often engaged by translating ribosomes. Moreover, they are encoded by genes that are significantly enriched in functions associated with transcriptional control, implying they may have an important and wide-ranging role in controlling cellular activities. VastDB thus provides an unprecedented resource for investigations of AS function and regulation.

PMID: 28855263 [PubMed - in process]



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Structural templating of J-aggregates: Visualizing bis(monoacylglycero)phosphate domains in live cells.

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Structural templating of J-aggregates: Visualizing bis(monoacylglycero)phosphate domains in live cells.

Biochim Biophys Acta. 2017 Nov;1865(11 Pt B):1687-1695

Authors: Mo GCH, Yip CM

Abstract
Identifying the key structural and dynamical determinants that drive the association of biomolecules, whether in solution, or perhaps more importantly in a membrane environment, has critical implications for our understanding of cellular dynamics, processes, and signaling. With recent advances in high-resolution imaging techniques, from the development of new molecular labels to technical advances in imaging methodologies and platforms, researchers are now reaping the benefits of being able to directly characterize and quantify local dynamics, structures, and conformations in live cells and tissues. These capabilities are providing unique insights into association stoichiometries, interactions, and structures on sub-micron length scales. We previously examined the role of lipid headgroup chemistry and phase state in guiding the formation of pseudoisocyanine (PIC) dye J-aggregates on supported planar bilayers [Langmuir, 25, 10719]. We describe here how these same J-aggregates can report on the in situ formation of organellar membrane domains in live cells. Live cell hyperspectral confocal microscopy using GFP-conjugated GTPase markers of early (Rab5) and late (Rab7) endosomes revealed that the PIC J-aggregates were confined to domains on either the limiting membrane or intralumenal vesicles (ILV) of late endosomes, known to be enriched in the anionic lipid bis(monoacylglycero)phosphate (BMP). Correlated confocal fluorescence - atomic force microscopy performed on endosomal membrane-mimetic supported planar lipid bilayers confirmed BMP-specific templating of the PIC J-aggregates. These data provide strong evidence for the formation of BMP-rich lipid domains during multivesicular body formation and portend the application of structured dye aggregates as markers of cellular membrane domain structure, size, and formation.

PMID: 28844737 [PubMed - in process]



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A Broad-Spectrum Inhibitor of CRISPR-Cas9.

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A Broad-Spectrum Inhibitor of CRISPR-Cas9.

Cell. 2017 Sep 07;170(6):1224-1233.e15

Authors: Harrington LB, Doxzen KW, Ma E, Liu JJ, Knott GJ, Edraki A, Garcia B, Amrani N, Chen JS, Cofsky JC, Kranzusch PJ, Sontheimer EJ, Davidson AR, Maxwell KL, Doudna JA

Abstract
CRISPR-Cas9 proteins function within bacterial immune systems to target and destroy invasive DNA and have been harnessed as a robust technology for genome editing. Small bacteriophage-encoded anti-CRISPR proteins (Acrs) can inactivate Cas9, providing an efficient off switch for Cas9-based applications. Here, we show that two Acrs, AcrIIC1 and AcrIIC3, inhibit Cas9 by distinct strategies. AcrIIC1 is a broad-spectrum Cas9 inhibitor that prevents DNA cutting by multiple divergent Cas9 orthologs through direct binding to the conserved HNH catalytic domain of Cas9. A crystal structure of an AcrIIC1-Cas9 HNH domain complex shows how AcrIIC1 traps Cas9 in a DNA-bound but catalytically inactive state. By contrast, AcrIIC3 blocks activity of a single Cas9 ortholog and induces Cas9 dimerization while preventing binding to the target DNA. These two orthogonal mechanisms allow for separate control of Cas9 target binding and cleavage and suggest applications to allow DNA binding while preventing DNA cutting by Cas9.

PMID: 28844692 [PubMed - indexed for MEDLINE]



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The Parkinson's disease-associated GPR37 receptor interacts with striatal adenosine A2A receptor controlling its cell surface expression and function in vivo.

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The Parkinson's disease-associated GPR37 receptor interacts with striatal adenosine A2A receptor controlling its cell surface expression and function in vivo.

Sci Rep. 2017 Aug 25;7(1):9452

Authors: Morató X, Luján R, López-Cano M, Gandía J, Stagljar I, Watanabe M, Cunha RA, Fernández-Dueñas V, Ciruela F

Abstract
G protein-coupled receptor 37 (GPR37) is an orphan receptor associated to Parkinson's disease (PD) neuropathology. Here, we identified GPR37 as an inhibitor of adenosine A2A receptor (A2AR) cell surface expression and function in vivo. In addition, we showed that GPR37 and A2AR do oligomerize in the striatum. Thus, a close proximity of GPR37 and A2AR at the postsynaptic level of striatal synapses was observed by double-labelling post-embedding immunogold detection. Indeed, the direct receptor-receptor interaction was further substantiated by proximity ligation in situ assay. Interestingly, GPR37 deletion promoted striatal A2AR cell surface expression that correlated well with an increased A2AR agonist-mediated cAMP accumulation, both in primary striatal neurons and nerve terminals. Furthermore, GPR37-/- mice showed enhanced A2AR agonist-induced catalepsy and an increased response to A2AR antagonist-mediated locomotor activity. Overall, these results revealed a key role for GPR37 controlling A2AR biology in the striatum, which may be relevant for PD management.

PMID: 28842709 [PubMed - in process]



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Taxonomically Restricted Genes with Essential Functions Frequently Play Roles in Chromosome Segregation in Caenorhabditis elegans and Saccharomyces cerevisiae.

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Taxonomically Restricted Genes with Essential Functions Frequently Play Roles in Chromosome Segregation in Caenorhabditis elegans and Saccharomyces cerevisiae.

G3 (Bethesda). 2017 Oct 05;7(10):3337-3347

Authors: Verster AJ, Styles EB, Mateo A, Derry WB, Andrews BJ, Fraser AG

Abstract
Genes encoding essential components of core cellular processes are typically highly conserved across eukaryotes. However, a small proportion of essential genes are highly taxonomically restricted; there appear to be no similar genes outside the genomes of highly related species. What are the functions of these poorly characterized taxonomically restricted genes (TRGs)? Systematic screens in Saccharomyces cerevisiae and Caenorhabditis elegans previously identified yeast or nematode TRGs that are essential for viability and we find that these genes share many molecular features, despite having no significant sequence similarity. Specifically, we find that those TRGs with essential phenotypes have an expression profile more similar to highly conserved genes, they have more protein-protein interactions and more protein disorder. Surprisingly, many TRGs play central roles in chromosome segregation; a core eukaryotic process. We thus find that genes that appear to be highly evolutionarily restricted do not necessarily play roles in species-specific biological functions but frequently play essential roles in core eukaryotic processes.

PMID: 28839119 [PubMed - in process]



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Genome-Scale Genetic Interactions and Cell Imaging Confirm Cytokinesis as Deleterious to Transient Topoisomerase II Deficiency in Saccharomyces cerevisiae.

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Genome-Scale Genetic Interactions and Cell Imaging Confirm Cytokinesis as Deleterious to Transient Topoisomerase II Deficiency in Saccharomyces cerevisiae.

G3 (Bethesda). 2017 Oct 05;7(10):3379-3391

Authors: Ramos-Pérez C, Ayra-Plasencia J, Matos-Perdomo E, Lisby M, Brown GW, Machín F

Abstract
Topoisomerase II (Top2) is an essential protein that resolves DNA catenations. When Top2 is inactivated, mitotic catastrophe results from massive entanglement of chromosomes. Top2 is also the target of many first-line anticancer drugs, the so-called Top2 poisons. Often, tumors become resistant to these drugs by acquiring hypomorphic mutations in the genes encoding Top2 Here, we have compared the cell cycle and nuclear segregation of two coisogenic Saccharomyces cerevisiae strains carrying top2 thermosensitive alleles that differ in their resistance to Top2 poisons: the broadly-used poison-sensitive top2-4 and the poison-resistant top2-5 Furthermore, we have performed genome-scale synthetic genetic array (SGA) analyses for both alleles under permissive conditions, chronic sublethal Top2 downregulation, and acute, yet transient, Top2 inactivation. We find that slowing down mitotic progression, especially at the time of execution of the mitotic exit network (MEN), protects against Top2 deficiency. In all conditions, genetic protection was stronger in top2-5; this correlated with cell biology experiments in this mutant, whereby we observed destabilization of both chromatin and ultrafine anaphase bridges by execution of MEN and cytokinesis. Interestingly, whereas transient inactivation of the critical MEN driver Cdc15 partly suppressed top2-5 lethality, this was not the case when earlier steps within anaphase were disrupted; i.e., top2-5 cdc14-1 We discuss the basis of this difference and suggest that accelerated progression through mitosis may be a therapeutic strategy to hypersensitize cancer cells carrying hypomorphic mutations in TOP2.

PMID: 28839115 [PubMed - in process]



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Data driven flexible backbone protein design.

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Data driven flexible backbone protein design.

PLoS Comput Biol. 2017 Aug;13(8):e1005722

Authors: Sun MGF, Kim PM

Abstract
Protein design remains an important problem in computational structural biology. Current computational protein design methods largely use physics-based methods, which make use of information from a single protein structure. This is despite the fact that multiple structures of many protein folds are now readily available in the PDB. While ensemble protein design methods can use multiple protein structures, they treat each structure independently. Here, we introduce a flexible backbone strategy, FlexiBaL-GP, which learns global protein backbone movements directly from multiple protein structures. FlexiBaL-GP uses the machine learning method of Gaussian Process Latent Variable Models to learn a lower dimensional representation of the protein coordinates that best represent backbone movements. These learned backbone movements are used to explore alternative protein backbones, while engineering a protein within a parallel tempered MCMC framework. Using the human ubiquitin-USP21 complex as a model we demonstrate that our design strategy outperforms current strategies for the interface design task of identifying tight binding ubiquitin variants for USP21.

PMID: 28837553 [PubMed - indexed for MEDLINE]



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Human Pluripotent Stem Cell-Derived TSC2-Haploinsufficient Smooth Muscle Cells Recapitulate Features of Lymphangioleiomyomatosis.

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Human Pluripotent Stem Cell-Derived TSC2-Haploinsufficient Smooth Muscle Cells Recapitulate Features of Lymphangioleiomyomatosis.

Cancer Res. 2017 Oct 15;77(20):5491-5502

Authors: Julian LM, Delaney SP, Wang Y, Goldberg AA, Doré C, Yockell-Lelièvre J, Tam RY, Giannikou K, McMurray F, Shoichet MS, Harper ME, Henske EP, Kwiatkowski DJ, Darling TN, Moss J, Kristof AS, Stanford WL

Abstract
Lymphangioleiomyomatosis (LAM) is a progressive destructive neoplasm of the lung associated with inactivating mutations in the TSC1 or TSC2 tumor suppressor genes. Cell or animal models that accurately reflect the pathology of LAM have been challenging to develop. Here, we generated a robust human cell model of LAM by reprogramming TSC2 mutation-bearing fibroblasts from a patient with both tuberous sclerosis complex (TSC) and LAM (TSC-LAM) into induced pluripotent stem cells (iPSC), followed by selection of cells that resemble those found in LAM tumors by unbiased in vivo differentiation. We established expandable cell lines under smooth muscle cell (SMC) growth conditions that retained a patient-specific genomic TSC2(+/-) mutation and recapitulated the molecular and functional characteristics of pulmonary LAM cells. These include multiple indicators of hyperactive mTORC1 signaling, presence of specific neural crest and SMC markers, expression of VEGF-D and female sex hormone receptors, reduced autophagy, and metabolic reprogramming. Intriguingly, the LAM-like features of these cells suggest that haploinsufficiency at the TSC2 locus contributes to LAM pathology, and demonstrated that iPSC reprogramming and SMC lineage differentiation of somatic patient cells with germline mutations was a viable approach to generate LAM-like cells. The patient-derived SMC lines we have developed thus represent a novel cellular model of LAM that can advance our understanding of disease pathogenesis and develop therapeutic strategies against LAM. Cancer Res; 77(20); 5491-502. ©2017 AACR.

PMID: 28830860 [PubMed - indexed for MEDLINE]



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Exercise-responsive phosphoproteins in the heart.

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Exercise-responsive phosphoproteins in the heart.

J Mol Cell Cardiol. 2017 Oct;111:61-68

Authors: Guo H, Isserlin R, Emili A, Burniston JG

Abstract
Endurance exercise improves cardiac performance and affords protection against cardiovascular diseases but the signalling events that mediate these benefits are largely unexplored. Phosphorylation is a widely studied post-translational modification involved in intracellular signalling, and to discover novel phosphorylation events associated with exercise we have profiled the cardiac phosphoproteome response to a standardised exercise test to peak oxygen uptake (VO2peak). Male Wistar rats (346±18g) were assigned to 3 independent groups (n=6, in each) that were familiarised with running on a motorised treadmill within a metabolic chamber. Animals performed a graded exercise test and were killed either immediately (0h) after or 3h after terminating the test at a standardised physiological end point (i.e. peak oxygen uptake; VO2peak). Control rats were killed at a similar time of day to the exercised animals, to minimise possible circadian effects. Cardiac proteins were digested with trypsin and phosphopeptides were enriched by selective binding to titanium dioxide (TiO2). Phosphopeptides were analysed by liquid chromatography and high-resolution tandem mass spectrometry, and phosphopeptides were quantified by MS1 intensities and identified against the UniProt knowledgebase using MaxQuant (data are available via ProteomeXchange, ID PXD006646). The VO2peak of rats in the 0h and 3h groups was 66±5mlkg(-1)min(-1) and 69.8±5mlkg(-1)min(-1), respectively. Proteome profiling detected 1169 phosphopeptides and one-way ANOVA found 141 significant (P<0.05 with a false discovery rate of 10%) differences. Almost all (97%) of the phosphosites that were responsive to exercise are annotated in the PhosphoSitePlus database but, importantly, the majority of these have not previously been associated with the cardiac response to exercise. More than two-thirds of the exercise-responsive phosphosites were different from those identified in previous phosphoproteome profiling of the cardiac response to β1-adrenergic receptor stimulation. Moreover, we report entirely new phosphorylation sites on 4 cardiac proteins, including S81 of muscle LIM protein, and identified 7 exercise-responsive kinases, including myofibrillar protein kinases such as obscurin, titin and the striated-muscle-specific serine/threonine kinase (SPEG) that may be worthwhile targets for future investigation.

PMID: 28826663 [PubMed - in process]



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Mechanism of bidirectional thermotaxis in Escherichia coli.

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Mechanism of bidirectional thermotaxis in Escherichia coli.

Elife. 2017 Aug 03;6:

Authors: Paulick A, Jakovljevic V, Zhang S, Erickstad M, Groisman A, Meir Y, Ryu WS, Wingreen NS, Sourjik V

Abstract
In bacteria various tactic responses are mediated by the same cellular pathway, but sensing of physical stimuli remains poorly understood. Here, we combine an in-vivo analysis of the pathway activity with a microfluidic taxis assay and mathematical modeling to investigate the thermotactic response of Escherichia coli. We show that in the absence of chemical attractants E. coli exhibits a steady thermophilic response, the magnitude of which decreases at higher temperatures. Adaptation of wild-type cells to high levels of chemoattractants sensed by only one of the major chemoreceptors leads to inversion of the thermotactic response at intermediate temperatures and bidirectional cell accumulation in a thermal gradient. A mathematical model can explain this behavior based on the saturation-dependent kinetics of adaptive receptor methylation. Lastly, we find that the preferred accumulation temperature corresponds to optimal growth in the presence of the chemoattractant serine, pointing to a physiological relevance of the observed thermotactic behavior.

PMID: 28826491 [PubMed - in process]



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