PaperPlayer biorxiv molecular biology

Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.08.09.213157v1?rss=1

Authors: Grimm, J., Shi, H., Wang, W., Mitchell, A. M., Wingreen, N. S., Huang, K. C., Silhavy, T. J.

Abstract:

The outer membrane (OM) of Gram-negative bacteria is a selective permeability barrier that allows uptake of nutrients while simultaneously protecting the cell from harmful compounds. The basic pathways and molecular machinery responsible for transporting lipopolysaccharides (LPS), lipoproteins, and {beta}-barrel proteins to the OM have been identified, but very little is known about phospholipid (PL) transport. To identify genes capable of affecting PL transport, we screened for genetic interactions with mlaA*, a mutant in which anterograde PL transport causes the inner membrane (IM) to shrink and eventually rupture; characterization of mlaA*-mediated lysis suggested that PL transport can occur via a high-flux, diffusive flow mechanism. We found that YhdP, an IM protein involved in maintaining the OM permeability barrier, modulates the rate of PL transport during mlaA*-mediated lysis. Deletion of yhdP from mlaA* reduced the rate of IM transport to the OM by 50%, slowing shrinkage of the IM and delaying lysis. As a result, the weakened OM of {triangleup}ydhP cells was further compromised and ruptured before the IM during mlaA*-mediated death. These findings demonstrate the existence of a high-flux, diffusive pathway for PL flow in Escherichia coli that is modulated by YhdP.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.08.10.243931v1?rss=1

Authors: Kara, N., Krueger, F., Rugg-Gunn, P., Houseley, J.

Abstract:

Understanding the distribution of sites at which replication forks stall, and the ensuing fork processing events, requires genome-wide methods sensitive to both changes in replication fork structure and the formation of recombinogenic DNA ends. Here we describe Tr ansferase- A ctivated E nd L igation seq uencing (TrAEL-seq), a method that captures single stranded DNA 3 ends genome-wide and with base pair resolution. TrAEL-seq labels DNA breaks, and profiles both stalled and processive replication forks in yeast and mammalian cells. Replication forks stalling at defined barriers and expressed genes are detected by TrAEL-seq with exceptional signal-to-noise, most likely through labelling of DNA 3 ends exposed during fork reversal. TrAEL-seq also labels unperturbed processive replication forks to yield maps of replication fork direction similar to those obtained by Okazaki fragment sequencing, however TrAEL-seq is performed on asynchronous populations of wild-type cells without incorporation of labels, cell sorting, or biochemical purification of replication intermediates, rendering TrAEL-seq simpler and more widely applicable than existing replication fork direction profiling methods. The specificity of TrAEL-seq for DNA 3 ends also allows accurate detection of double strand break sites after the initiation of DNA end resection, which we demonstrate by genome-wide mapping of meiotic double strand break hotspots in a dmc1 {Delta} mutant. Overall, TrAEL-seq provides a flexible and robust methodology with high sensitivity and resolution for studying DNA replication and repair, which will be of significant use in determining mechanisms of genome instability.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.08.10.245076v1?rss=1

Authors: Shibata, E., Dutta, A.

Abstract:

The six-subunit Origin Recognition Complex (ORC) binds to origins of replication as a ring-shaped heterohexameric ATPase that is essential to recruit MCM2-7 and initiate DNA replication. We now report that human HCT116 colon cancer cells survive with a mutation in the initiator ATG of the ORC5 gene that abolishes the expression of ORC5 protein. Even if an internal methionine is used to produce an undetectable, N terminally deleted ORC5, the protein would lack 80% of the AAA+ ATPase domain, including the Walker A motif. The ORC5-depleted cells show normal chromatin binding of MCM2-7 and initiate replication from similar number of origins as wild type cells. A second mutation in ORC2 in ORC5 mutant cells rendered both ORC5 and ORC2 proteins undetectable, and yet the cells grow, recruit MCM2-7 normally to chromatin and initiate DNA replication with similar number of origins. Thus, either a crippled ORC lacking ORC2 and ORC5 can load MCM2-7 to initiate DNA replication, or human cell-lines can recruit MCM2-7 to origins in a reaction independent of ORC.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.08.10.243170v1?rss=1

Authors: Lozano-Vidal, N., Stanicek, L., Bink, D. I., Kremer, V., MacInnes, A. W., Dimmeler, S., Boon, R. A.

Abstract:

Age-related diseases pose great challenges to health care systems worldwide. During aging, endothelial senescence increases the risk for cardiovascular disease. Recently, it was described that Phosphatase 1 Nuclear Targeting Subunit (PNUTS) has a central role in cardiomyocyte aging and homeostasis. Here, we determined the role of PNUTS in endothelial cell aging. We confirmed that PNUTS is repressed in senescent endothelial cells (ECs). Moreover, PNUTS silencing elicits several of the hallmarks of endothelial aging: senescence, reduced angiogenesis and loss of barrier function. To validate our findings in vivo, we generated an endothelial-specific inducible PNUTS-deficient mouse line (Cdh5-CreERT2;PNUTSfl/fl), termed PNUTSEC-KO. Two weeks after PNUTS deletion, PNUTSEC-KO mice presented severe multiorgan failure and vascular leakage. We showed that the PNUTS binding motif for protein phosphatase 1 (PP1) is essential to maintain endothelial barrier function. Transcriptomic analysis of PNUTS-silenced HUVECs and lungs of PNUTSEC-KO mice revealed that the PNUTS-PP1 axis tightly regulates the expression of semaphorin 3B (SEMA3B). Indeed, silencing of SEMA3B completely restored barrier function after PNUTS loss-of-function. These results reveal a pivotal role for PNUTS in endothelial homeostasis through a PP1-SEMA3B downstream pathway that provides a potential target against the effects of aging in ECs.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.08.09.243451v1?rss=1

Authors: Yuan, S., Peng, L., Park, J. J., Hu, Y., Devarkar, S. C., Dong, M. B., Wu, S., Chen, S., Lomakin, I., Xiong, Y.

Abstract:

The COVID-19 pandemic affects millions of people worldwide with a rising death toll. The causative agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), uses its nonstructural protein 1 (Nsp1) to redirect host translation machinery to the viral RNA by binding to the ribosome and suppressing cellular, but not viral, protein synthesis through yet unknown mechanisms. We show here that among all viral proteins, Nsp1 has the largest impact on host viability in the cells of human lung origin. Differential expression analysis of mRNA-seq data revealed that Nsp1 broadly alters the transcriptome in human cells. The changes include repression of major gene clusters in ribosomal RNA processing, translation, mitochondria function, cell cycle and antigen presentation; and induction of factors in transcriptional regulation. We further gained a mechanistic understanding of the Nsp1 function by determining the cryo-EM structure of the Nsp1-40S ribosomal subunit complex, which shows that Nsp1 inhibits translation by plugging the mRNA entry channel of the 40S. We also determined the cryo-EM structure of the 48S preinitiation complex (PIC) formed by Nsp1, 40S, and the cricket paralysis virus (CrPV) internal ribosome entry site (IRES) RNA, which shows that this 48S PIC is nonfunctional due to the incorrect position of the 3' region of the mRNA. Results presented here elucidate the mechanism of host translation inhibition by SARS-CoV-2, provide insight into viral protein synthesis, and furnish a comprehensive understanding of the impacts from one of the most potent pathogenicity factors of SARS-CoV-2.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.08.07.240846v1?rss=1

Authors: Choe, H. M., Luo, Z.-B., Xuan, M.-F., Quan, B.-H., Kang, J.-D., Oh, M. J., An, H. J., Yin, X.-J.

Abstract:

The recent GGTA1 and CMAH DKO pigs have made it possible to resolve the immune barriers which are duo to xenoantigens on RBC such as Gal and Neu5Gc. Nevertheless, it still requires the detection of glycosylation alternation on the pig RBCs because even the minor changes would be unexpected xenoantigens. DKO RBC immune reactivity with human serum was assessed by hemagglutination assay. Glycosylation alteration of RBC membranes was characterized by NanoLC-Q-TOF-MS system and lectin blotting assay. Twelve GGTA1/CMAH DKO piglets were successfully produced. The immunoreactivity with human serum was remarkably reduced in DKO (less than 1:2 dilution), whereas wild type(WT) pigs showed agglutination (the least 1:256 dilution). The MS results showed that DKO increased neutral N-glycans as well as decreased total sialylated N-glycans, especially suggesting significant decrease of di-sialylated N-glycans (P < 0.05). Moreover, lectin blotting assay revealed that DKO pigs reduced the binding signals with AAL, AOL, LCA and SNA and increased the binding signal with MAL. DKO pigs decreased the expression of total fucosylation and sialylated N-glycans on the erythrocyte membrane. Our findings will support further investigation into DKO pig RBC glycosylation and contribute to uncover the roles of glycan changes for xenotransfusion.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.08.05.237479v1?rss=1

Authors: Sun, Q.

Abstract:

CRM1 is an important drug target in diseases such as cancer and viral infection. Plumbagin and oridonin, the herbal ingredients with known anti-cancer activities, were reported to inhibit CRM1-mediated nuclear export. However, their modes of CRM1 inhibition are unclear. Here, a multi-mutant of yeast CRM1 was engineered to enable the crystallization of these two small molecules in CRM1's NES-binding groove. Each structure showed three inhibitor-binding sites, among which two are conserved in humans. Besides the known binding site, another site also participated in oridonin and plumbagin's CRM1 inhibition. While the plumbagin-bound NES groove resembled the NES-bound groove state, the oridonin-bound groove revealed for the first time a more open NES groove, which may potentially improve cargo-loading through a capture-and-tighten mechanism. Our work thus provides a tool for CRM1 inhibitor crystallization, new insights of CRM1-cargo interaction, and a structural basis for further development of these or other CRM1 inhibitors.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.08.06.239384v1?rss=1

Authors: Tanneti, S. N., Joel D Federspiel, J. D., Ileana M Cristea, I. M., Enquist, L. W.

Abstract:

Alpha-herpesviruses establish a life-long infection in the nervous system of the affected host; while this infection is restricted to peripheral neurons in a healthy host, the reactivated virus can spread within the neuronal circuitry, such as to the brain, in compromised individuals and lead to adverse health outcomes. Pseudorabies virus (PRV), an alpha-herpesvirus, requires the viral protein Us9 to sort virus particles into axons and facilitate neuronal spread. Us9 sorts virus particles by mediating the interaction of virus particles with neuronal transport machinery. Here, we report that Us9-mediated regulation of axonal sorting also depends on the state of neuronal maturation. Specifically, the development of dendrites and axons is accompanied with proteomic changes that influence neuronal processes. Immature superior cervical ganglionic neurons (SCGs) have rudimentary neurites that lack markers of mature axons. Immature SCGs can be infected by PRV, but they show markedly reduced Us9-dependent regulation of sorting, and increased Us9-independent transport of particles into neurites. Mature SCGs have relatively higher abundances of proteins characteristic of vesicle-transport machinery. We also identify Us9-associated neuronal proteins that can contribute to axonal sorting and subsequent anterograde spread of virus particles in axons. We show that SMPD4/nsMase3, a sphingomyelinase abundant in lipid-rafts, associates with Us9 and is a negative regulator of PRV sorting into axons and neuronal spread, a potential antiviral function.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.08.06.240358v1?rss=1

Authors: Chou, H.-C., Bhalla, K., Demerdesh, O. E., Klingbeil, O., Hanington, K., Aganezov, S., Andrews, P., Alsudani, H., Chang, K., Vakoc, C. R., Schatz, M. C., McCombie, W. R., Stillman, B.

Abstract:

The Origin Recognition Complex (ORC) cooperates with CDC6, MCM2-7, and CDT1 to form pre-RC complexes at origins of DNA replication. Here we report tiling-sgRNA CRISPR screens that show that each subunit of ORC and CDC6 are essential in human cells. Using an auxin-inducible degradation system, stable cell lines were created that ablate ORC2 rapidly, revealing multiple cell division cycle phenotypes. The primary defect in the absence of ORC2 was cells encountering difficulty in initiating DNA replication or progressing through the cell division cycle due to reduced MCM2-7 loading onto chromatin in G1 phase. The nuclei of ORC2 deficient cells were also large, with decompacted heterochromatin. Some ORC2 deficient cells that completed DNA replication entered into, but never exited mitosis. ORC1 knockout cells also demonstrated extremely slow cell proliferation and abnormal cell and nuclear morphology. Thus, ORC proteins and CDC6 are indispensable for normal cellular proliferation and contribute to nuclear organization.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.08.06.239723v1?rss=1

Authors: Aida, T., Wilde, J. J., Yang, L., Hou, Y., Li, M., Xu, D., Lin, J., Qi, P., Lu, Z., Feng, G.

Abstract:

Genome editing has transformed biomedical science, but is still unpredictable and often induces undesired outcomes. Prime editing (PE) is a promising new approach due to its proposed flexibility and ability to avoid unwanted indels. Here, we show highly efficient PE-mediated genome editing in mammalian zygotes. Utilizing chemically modified guideRNAs, PE efficiently introduced 10 targeted modifications including substitutions, deletions, and insertions across 6 genes in mouse embryos. However, we unexpectedly observed a high frequency of undesired outcomes such as large deletions and found that these occurred more often than pure intended edits across all of the edits/genes. We show that undesired outcomes result from the doublenicking PE3 strategy, but that omission of the second nick largely ablates PE function. However, sequential double-nicking with PE3b, which is only applicable to a fraction of edits, eliminated undesired outcomes. Overall, our findings demonstrate the promising potential of PE for predictable, flexible, and highly efficient in vivo genome editing, but highlight the need for improved variations of PE before it is ready for widespread use.

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