PaperPlayer biorxiv molecular biology

Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.07.22.216804v1?rss=1

Authors: Wei, H., Movahedi, A., Xu, C., Sun, W., Wang, P., Li, D., Zhuge, Q.

Abstract:

Plants use two distinct isoprenoid biosynthesis pathways: the methylerythritol phosphate (MEP) pathway and mevalonic acid (MVA) pathway. 1-deoxy-D-xylulose5-phosphate synthase (DXS) and 1-deoxy-D-xylulose5-phosphate reductoisomerase (DXR) are the rate-limiting enzymes in the MEP pathway, and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) is a key regulatory enzyme in the MVA pathway. Previously, overexpression of Populus trichocarpa PtDXR in Nanlin 895 poplar was found to upregulate resistance to salt and drought stresses, and the transgenic poplars showed improved growth. In the present study, PtHMGR overexpressors (OEs) exhibited higher expression levels of DXS, DXR, 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase (HDS), and 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase (HDR) and lower expression levels of 2-C-methyl-d-erythritol4-phosphate cytidylyltransferase (MCT), 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (CMK), and 3-hydroxy-3-methylglutaryl-coenzyme A synthase (HMGS) than non-transgenic poplars (NT). However, the poplar PtDXR-OEs showed upregulated expression levels of MEP-related genes and downregulated expression of MVA-related genes. Moreover, overexpression of PtDXR and PtHMGR in poplars caused changes in MVA-derived trans-zeatin-riboside (TZR), isopentenyl adenosine (IPA), castasterone (CS) and 6-deoxocastasterone (DCS), as well as MEP-derived carotenoids, gibberellins (GAs), and abscisic acid (ABA). In PtHMGR-OEs, greater accumulation of geranyl diphosphate synthase (GPS) and geranyl pyrophosphate synthase (GPPS) transcript levels in the MEP pathway led to accumulation of MEP-derived isoprenoids, while upregulation of farnesyl diphosphate synthase (FPS) expression in the MVA pathway contributed to increased levels of MVA-derived isoprenoids. Similarly, in PtDXR-OEs, increased GPS and GPPS transcript levels in the MEP pathway boosted MEP-derived isoprenoid levels and changes in FPS expression affected MVA-derived isoprenoid yields. From these results, we can conclude that cross-talk exists between the MVA and MEP pathways.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.07.19.210997v1?rss=1

Authors: Gollavilli, P. N., Parma, B., Siddiqui, M. A., Yang, H., Ramesh, V., Napoli, F., Schwab, A., Natesan, R., Asangani, I. A., Brabletz, T., Pilarsky, C., Ceppi, P.

Abstract:

Since their discovery, microRNAs (miRNA)s have been widely studied in almost every aspect of biology and medicine, leading to the identification of important gene regulation circuits and cellular mechanisms. However, investigations are generally focused on the analysis of their downstream targets and biological functions in overexpression and knockdown approaches, while miRNAs endogenous levels and activity remain poorly understood. Here, we used the cellular plasticity-regulating process of epithelial-to-mesenchymal transition (EMT) as a model to show the efficacy of a fluorescent sensor to separate cells with distinct EMT signatures, based on miR-200b/c activity. The system was further combined with a CRISPR-Cas9 screening platform to unbiasedly identify miR-200b/c upstream regulating genes. The sensor allows to infer miRNAs fundamental biological properties, as profiling of sorted cells indicated miR-200b/c as a molecular switch between EMT differentiation and proliferation, and suggested a role for metabolic enzymes in miR-200/EMT regulation. Analysis of miRNAs endogenous levels and activity could lead to a better understanding of their biological role in physiology and disease.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.07.21.175117v1?rss=1

Authors: Yates, J. D., Russell, R. C., Yost, H. J., Hill, J. T.

Abstract:

CRISPR-Cas9 sgRNA libraries have transformed functional genetic screening and have enabled innovative CRISPR-based methods, such as the visualization of chromatin dynamics in living cells. These libraries have the potential to be applied to a vast number of biological systems and aid in the development of new technologies, but their synthesis is hindered by the cost, time requirements, and technical difficulty of current sgRNA library generation methods. Here, we describe SLALOM--a rapid enzymatic method for generating robust, variant-matched sgRNA libraries from any source of DNA in under 3 hours. This method utilizes a custom sgRNA scaffold sequence and a novel method for detaching oligonucleotides from solid supports using a strand displacing polymerase. Using this method, we have constructed libraries targeting the E. coli genome and the transcriptome of developing zebrafish hearts, demonstrating its potential to expand the reach of CRISPR technology and facilitate methods requiring custom sgRNA libraries.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.07.20.211417v1?rss=1

Authors: Zhang, Y., Xiong, X., Zhu, Q., Zhang, J., Wang, Y., Cao, J., Chen, L., Chen, S., Hou, L., Zhao, X., Hao, P., Zhuang, M., Li, D., Fan, G.

Abstract:

Tyrosine phosphorylation, orchestrated by tyrosine kinases and phosphatases, modulates a multi-layered signaling network in a time and space dependent manner. Dysregulation of this post-translational modification is inevitably associated with pathological diseases. Our previous work has demonstrated that non-receptor tyrosine kinase FER is upregulated in ovarian cancer. Knockdown of the kinase attenuates metastatic phenotypes in tumor cells. Here we employed mass spectrometry and biochemical approaches to identify IRS4 as a novel substrate of FER. Using a proximity-based tagging system, we determined that FER-mediated phosphorylation of Tyr779 enables IRS4 to recruit PIK3R2/p85{beta}, the regulatory subunit of PI-3K, and activate the PI3K-AKT pathway. Rescuing IRS4-null ovarian tumor cells with phosphorylation-defective mutant, but not WT IRS4, delayed tumor cell proliferation both in vitro and in vivo. Overall, we revealed a kinase-substrate regulatory mode between FER and IRS4, and the pharmacological inhibition of FER kinase may be beneficial for ovarian cancer patients with PI3K-AKT hyperactivation.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.07.20.213066v1?rss=1

Authors: Pleiner, T., Januszyk, K., Tomaleri, G. P., Oania, R. S., Hazu, M., Sweredoski, M. J., Moradian, A., Guna, A., Voorhees, R. M.

Abstract:

The assembly of nascent proteins into multi-subunit complexes is tightly regulated to maintain cellular homeostasis. The ER membrane protein complex (EMC) is an essential insertase that requires seven membrane-spanning and two soluble subunits for function. Here we show that the kinase With no lysine 1 (WNK1), known for its role in hypertension and neuropathy, is required for assembly of the human EMC. WNK1 uses a conserved amphipathic helix to stabilize the soluble subunit, EMC2, by binding to the EMC2-8 interface. Shielding this hydrophobic surface prevents promiscuous interactions of unassembled EMC2 and precludes binding of ubiquitin ligases, permitting assembly. Using biochemical reconstitution, we show that after EMC2 reaches the membrane, its interaction partners within the EMC displace WNK1, and similarly shield its exposed hydrophobic surfaces. This work describes an unexpected role for WNK1 in protein biogenesis, and defines the general requirements of an assembly factor that will apply across the proteome.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.07.20.212050v1?rss=1

Authors: Claycomb, J. M., Charlesworth, A. G., Lehrbach, N. J., Seroussi, U., Renaud, M. S., Molnar, R. I., Woock, J. R., Aber, M. J., Diao, A. J., Ruvkun, G.

Abstract:

The C. elegans genome encodes nineteen functional Argonaute proteins that utilize 22G-RNAs, 26G-RNAs, miRNAs, or piRNAs to regulate their target transcripts. Only one of these proteins is essential under normal laboratory conditions: CSR-1. While CSR-1 has been studied in various developmental and functional contexts, nearly all studies investigating CSR-1 have overlooked the fact that the csr-1 locus encodes two isoforms. These isoforms differ by an additional 163 amino acids present in the N-terminus of CSR-1a. Using CRISPR-Cas9 genome editing to introduce GFP::3xFLAG epitopes into the long (CSR-1a) and short (CSR-1b) isoforms of CSR-1, we identified differential expression patterns for the two isoforms. CSR-1a is expressed specifically during spermatogenesis and in select somatic tissues, including the intestine. In contrast, CSR-1b, is expressed constitutively in the germline. Essential functions of csr-1 described in the literature coincide with CSR-1b. In contrast, CSR-1a plays tissue specific functions during spermatogenesis, where it integrates into a spermatogenesis sRNA regulatory network including ALG-3, ALG-4, and WAGO-10 that is necessary for male fertility. CSR-1a is also required in the intestine for the silencing of repetitive transgenes. Sequencing of small RNAs associated with each CSR-1 isoform reveals that CSR-1a engages with 22G- and 26G-RNAs, while CSR-1b interacts with only 22G-RNAs to regulate distinct groups of germline genes and regulate both sperm and oocyte-mediated fertility.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.07.20.213108v1?rss=1

Authors: Tsang, H.-G., Lillico, S., Proudfoot, C., McCulloch, M. E. B., Markby, G. R., Trejo-Reveles, V., Whitelaw, C. B. A., MacRae, V. E., Summers, K. M.

Abstract:

This paper describes a genome editing project using CRISPR-Cas9. The objective was to create a large animal model of human Marfan syndrome by targeting the FBN1 gene of the pig, Sus scrofa, using a single guide and non-homologous end joining which was expected to create short insertion or deletion mutations at the 5 end of the gene. The editing successfully created a five base pair deletion in exon 2 of FBN1, which was homozygous in two animals. However, the phenotype of these piglets was unexpected, since they showed none of the signs consistent with Marfan syndrome but both suffered extreme hydrops fetalis with a large amount of fluid located under the skin and in the abdomen. One of the edited piglets was stillborn and the other was euthanised at birth on welfare grounds. It is likely that this result was due to unanticipated on- or off-target mutations, possibly in the GLDN gene 3 megabases away from FBN1. This result provides more evidence for unexpected outcomes of CRISPR- Cas9 gene editing and supports the proposal that all genome edited individuals should be subjected to strategies to track the CRISPR footprint, such as whole genome sequencing, before being used for further experimentation or in clinical applications.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.07.21.214338v1?rss=1

Authors: Sheahan, T. W., Major, V., Webb, K. M., Bryan, E., Voigt, P.

Abstract:

The closely related acetyltransferases CBP and p300 are key regulators of gene expression in metazoans. CBP/p300 acetylate several specific lysine residues within nucleosomes, including histone H3 lysine 27 (H3K27), a hallmark of active enhancers and promoters. However, it has remained largely unclear how specificity of CBP/p300 towards H3K27 is achieved. Here we show that the TAZ2 domain of CBP is required for efficient acetylation of H3K27, while curbing activity towards other lysine residues within nucleosomes. We find that TAZ2 is a sequence-independent DNA binding module, promoting interaction between CBP and nucleosomes, thereby enhancing enzymatic activity and regulating substrate specificity of CBP. TAZ2 is further required to stabilize CBP binding to chromatin in mouse embryonic stem cells, facilitating specificity towards H3K27 and modulating gene expression. These findings reveal a crucial role of TAZ2 in regulating H3K27ac, while highlighting the importance of correct site-specific acetylation for proper regulation of gene expression.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.07.20.212290v1?rss=1

Authors: Loghry, H. J., Yuan, W., Zamanian, M., Wheeler, N. J., Day, T. A., Kimber, M. J.

Abstract:

Lymphatic filariasis (LF) is a disease caused by parasitic filarial nematodes that is endemic in 49 countries and affects or threatens over 890 million people. Strategies to control LF rely heavily on mass administration of anthelmintic drugs including ivermectin (IVM), a macrocyclic lactone drug considered an Essential Medicine by the WHO. However, despite its widespread use the therapeutic mode of action of IVM against filarial nematodes is not clear. We have previously reported that filarial nematodes secrete extracellular vesicles (EVs) and that their cargo has immunomodulatory properties. Here we investigate the effects of IVM and other anti-filarial drugs on parasitic nematode EV secretion, motility, and protein secretion. We show that inhibition of EV secretion was a specific property of IVM, which had consistent and significant inhibitory effects across nematode life stages and species (with the exception of male parasites). IVM inhibited EV secretion, but not parasite motility, at therapeutically relevant concentrations. Protein secretion was inhibited by IVM in the microfilariae stage, but not in any other stage tested. Our data provides evidence that inhibiting the secretion of immunomodulatory EVs by parasitic nematodes could explain, at least in part, IVM mode of action and provides a phenotype for novel drug discovery.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.07.21.213546v1?rss=1

Authors: wang, t.

Abstract:

Both pathogenesis and detection of liver fibrosis and early cirrhosis are elusive. Our study focused on role of microRNAs (miRNAs) as predictive biomarkers of liver fibrosis and early cirrhosis. Methods: We recruited patients with chronic hepatitis B background and divided them into three subgroups according to the pathological staging results: no liver fibrosis group (CHB 11, 12.0%), liver fibrosis group but without liver cirrhosis (LF 41, 44.6%), and early liver cirrhosis group (LC 40, 43.4%). Results: Levels of miR-122 in the CHB and LF group were significantly elevated compared with the LC group. Meanwhile, upregulation levels of miR-146a-5p, miR-29c-3p, miR-223 were seen in the CHB group versus LC. MiR-122-5p was best able to differentiate between patients with LC and without LC (area under the curve (AUC) 0.734). It can also distinguish patients with LF from those without LF (AUC 0.681). Panel including miR-122-5p miR-29c-3p and miR-381-3p resulted in an AUC of 0.700 in detection of LF. Meanwhile panel including miR-122-5p, miR-29c-3p, MiR-223 turned out to be good predictors, resulted in an AUC of 0.752 to discriminate LC in HBV-associated disease. HBV load, ALT and AST showed significant positive correlation with miR-122-5p and miR-381-3p. Conclusions: Our study explored changes of plasm miRNA levels in HBV-associated disease patients, constructed diagnostic miRNAs panel to predict liver fibrosis and early cirrhosis. Meanwhile In the process of liver fibrosis and cirrhosis, there are involving not only common pathway molecules, but also its specific pathway molecules.

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