PaperPlayer biorxiv molecular biology

Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.08.12.247825v1?rss=1

Authors: Wang, Y., Fan, Y., Huang, Y., Du, T., Liu, Z., Huang, D., Wang, Y., Wang, N., Zhang, P.

Abstract:

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of coronavirus disease 2019 (COVID-19), it binds to angiotensin-converting enzyme 2 (ACE2) to enter into human cells. The expression level of ACE2 potentially determine the susceptibility and severity of COVID-19, it is thus of importance to understand the regulatory mechanism of ACE2 expression. Tripartite motif containing 28 (TRIM28) is known to be involved in multiple processes including antiviral restriction, endogenous retrovirus latency and immune response, it is recently reported to be co-expressed with SARS-CoV-2 receptor in type II pneumocytes; however, the roles of TRIM28 in ACE2 expression and SARS-CoV-2 cell entry remain unclear. This study showed that knockdown of TRIM28 induces ACE2 expression and increases pseudotyped SARS-CoV-2 cell entry of A549 cells and primary pulmonary alveolar epithelial cells (PAEpiCs). In a co-culture model of NK cells and lung epithelial cells, our results demonstrated that NK cells inhibit TRIM28 and promote ACE2 expression in lung epithelial cells, which was partially reversed by depletion of interleukin-2 and blocking of granzyme B in the co-culture medium. Furthermore, TRIM28 knockdown enhanced interferon-{gamma} (IFN-{gamma})-induced ACE2 expression through a mechanism involving upregulating IFN-{gamma} receptor 2 (IFNGR2) in both A549 and PAEpiCs. Importantly, the upregulated ACE2 induced by TRIM28 knockdown and co-culture of NK cells was partially reversed by dexamethasone in A549 cells but not PAEpiCs. Our study identified TRIM28 as a novel regulator of ACE2 expression and SARS-CoV-2 cell entry.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.08.12.225458v1?rss=1

Authors: Yarani, R., Palasca, O., Doncheva, N. T., Anthon, C., Pilecki, B., Svane, C. A. S., Mirza, A. H., Litman, T., Holmskov, U., Bang-Berthelsen, C. H., Vilien, M., Jensen, L. J., Gorodkin, J., Pociot, F.

Abstract:

BACKGROUND & AIMS: Ulcerative colitis (UC) is an inflammatory bowel disorder with unknown etiology. Given its complex nature, in vivo studies to investigate its pathophysiology is vital. Animal models play an important role in molecular profiling necessary to pinpoint mechanisms that contribute to human disease. Thus, we aim to identify common conserved gene expression signatures and differentially regulated pathways between human UC and a mouse model hereof, which can be used to identify UC patients from healthy individuals and to suggest novel treatment targets and biomarker candidates. METHODS: Therefore, we performed high-throughput total and small RNA sequencing to comprehensively characterize the transcriptome landscape of the most widely used UC mouse model, the dextran sodium sulfate (DSS) model. We used this data in conjunction with publicly available human UC transcriptome data to compare gene expression profiles and pathways. RESULTS: We identified differentially regulated protein-coding genes, long non-coding RNAs and microRNAs from colon and blood of UC mice and further characterized the involved pathways and biological processes through which these genes may contribute to disease development and progression. By integrating human and mouse UC datasets, we suggest a set of 51 differentially regulated genes in UC colon and blood that may improve molecular phenotyping and aid in treatment decisions, drug discovery and the design of clinical trials. CONCLUSION: Global transcriptome analysis of the DSS-UC mouse model supports its use as an efficient high-throughput tool to discover new targets for therapeutic and diagnostic applications in human UC through identifying relationships between gene expression and disease phenotype. KEYWORDS: DSS; UC; Transcriptomics; Colon; Blood

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.08.11.242081v1?rss=1

Authors: Arrastia, M. V., Jachowicz, J. W., Ollikainen, N., Curtis, M. S., Lai, C., Quinodoz, S., Selck, D. A., Guttman, M., Ismagilov, R. F.

Abstract:

In eukaryotes, the nucleus is organized into a three dimensional structure consisting of both local interactions such as those between enhancers and promoters, and long-range higher-order structures such as nuclear bodies. This organization is central to many aspects of nuclear function, including DNA replication, transcription, and cell cycle progression. Nuclear structure intrinsically occurs within single cells; however, measuring such a broad spectrum of 3D DNA interactions on a genome-wide scale and at the single cell level has been a great challenge. To address this, we developed single-cell split-pool recognition of interactions by tag extension (scSPRITE), a new method that enables measurements of genome-wide maps of 3D DNA structure in thousands of individual nuclei. scSPRITE maximizes the number of DNA contacts detected per cell enabling high-resolution genome structure maps within each cells and is easy-to-use and cost-effective. scSPRITE accurately detects chromosome territories, active and inactive compartments, topologically associating domains (TADs), and higher-order structures within single cells. In addition, scSPRITE measures cell-to-cell heterogeneity in genome structure at different levels of resolution and shows that TADs are dynamic units of genome organization that can vary between different cells within a population. scSPRITE will improve our understanding of nuclear architecture and its relationship to nuclear function within an individual nucleus from complex cell types and tissues containing a diverse population of cells.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.08.11.245068v1?rss=1

Authors: Hill, C. H., Cook, G., Napthine, S., Kibe, A., Brown, K., Caliskan, N., Firth, A. E., Graham, S. C., Brierley, I.

Abstract:

The 2A protein of Theiler's murine encephalomyelitis virus (TMEV) is required for stimulating programmed -1 ribosomal frameshifting (PRF) during infection. However, the amino acid sequence of TMEV 2A shares only 27% identity with the 2A orthologue from the related cardiovirus encephalomyocarditis virus (EMCV) for which a structure has been recently determined. Here we present the X-ray crystal structure of TMEV 2A, revealing that, despite the low sequence identity, the overall beta-shell architecture is retained, and the location of previously described mutations on this structure suggests a common RNA binding mode. We determine the minimal stimulatory element in the viral RNA required for 2A binding and show that 2A binds to this element with 1:1 stoichiometry and nanomolar affinity. We also demonstrate a critical role for bases upstream of the originally predicted stem-loop, providing evidence that an alternative pseudoknot-like conformation recently demonstrated for EMCV is a conserved feature of cardiovirus stimulatory elements. We go on to examine frameshifting in infected cells by ribosome profiling and metabolic labelling. We observe PRF efficiencies of up to 85%, highlighting this as the most efficient example of -1 PRF in any natural system thus far characterised. Furthermore, we document a series of ribosomal pauses in and around the site of PRF with potential implications for our understanding of translational control in cardioviruses.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.08.11.245035v1?rss=1

Authors: Hill, C. H., Napthine, S., Pekarek, L., Kibe, A., Firth, A. E., Graham, S. C., Caliskan, N., Brierley, I.

Abstract:

Encephalomyocarditis virus 2A protein is a multi functional virulence factor essential for efficient virus replication with roles in stimulating programmed -1 ribosomal frameshifting (PRF), inhibiting cap-dependent translational initiation, interfering with nuclear import and export and preventing apoptosis of infected cells. The mechanistic basis for many of these activities is unclear and a lack of structural data has hampered our understanding. Here we present the X-ray crystal structure of 2A, revealing a novel 'beta-shell' fold. We show that 2A selectively binds to and stabilises a specific conformation of the stimulatory RNA element in the viral genome that directs PRF at the 2A/2B* junction. We dissect the folding energy landscape of this stimulatory RNA element, revealing multiple conformers, and measure changes in unfolding pathways arising from mutation and 2A binding. Furthermore, we demonstrate a strong interaction between 2A and the small ribosomal subunit and present a high-resolution cryo-EM structure of 2A bound to initiated 70S ribosomes. In this complex, three copies of 2A bind directly to 16S ribosomal RNA at the factor binding site, where they may compete for binding with initiation and elongation factors. Together, these results provide an integrated view of the structural basis for RNA recognition by 2A, expand our understanding of PRF, and provide unexpected insights into how a multifunctional viral protein may shut down translation during virus infection.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.08.11.246231v1?rss=1

Authors: Wassing, I. E., Saayman, X., Rampazzo, L., Ralf, C., Bassett, A., Esashi, F.

Abstract:

The RAD51 recombinase plays critical roles in safeguarding genome integrity, which is fundamentally important for all living cells. While interphase functions of RAD51 in repairing broken DNA and protecting stalled replication forks are well characterised, its role in mitosis remains contentious. In this study, we show that RAD51 protects under-replicated DNA in mitotic human cells and, in this way, promotes mitotic DNA synthesis (MiDAS) and successful chromosome segregation. MiDAS was globally detectable irrespective of DNA damage and was promoted by de novo RAD51 recruitment, RAD51-mediated fork protection, and RAD51 phosphorylation by the key mitotic regulator Polo-like kinase 1. Importantly, acute inhibition of RAD51-promoted MiDAS led to mitotic DNA damage, delayed anaphase onset and induced centromere fragility, revealing a mechanism that prevents the satisfaction of the spindle assembly checkpoint when chromosomal replication remains incomplete. This study hence identifies an unexpected function of RAD51 in promoting the stability of mitotic chromatin.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.08.11.244855v1?rss=1

Authors: Brodolin, K., Morichaud, Z.

Abstract:

The basal transcription factors of cellular RNA polymerases (RNAPs) stimulate the initial RNA synthesis via poorly understood mechanisms. Here, we explored the mechanism employed by the bacterial factor {sigma} in promoter-independent initial transcription. We found that the RNAP holoenzyme lacking the promoter-binding domain {sigma}4 is ineffective in de novo transcription initiation and displays high propensity to pausing upon extension of RNAs 3 to 7 nucleotides in length. The {sigma}4 domain stabilizes short RNA:DNA hybrids and suppresses pausing by stimulating RNAP active-center translocation. The anti-pausing activity of {sigma}4 is modulated by its interaction with the {beta} subunit flap domain and by the {sigma} remodeling factors AsiA and RbpA. Our results suggest that the presence of {sigma}4 within the RNA exit channel compensates for the intrinsic instability of short RNA:DNA hybrids by increasing RNAP processivity, thus favoring productive transcription initiation. This RNAP boosting activity of the initiation factor is shaped by the the thermodynamics of RNA:DNA interactions and thus, should be relevant for any factor-dependent RNAP.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.08.11.246249v1?rss=1

Authors: Pashler, A. L., Towler, B. P., Jones, C. I., Burgess, T., Newbury, S. F.

Abstract:

XRN1 is a highly conserved exoribonuclease which degrades uncapped RNAs in a 5'-3' direction. Degradation of RNAs by XRN1 is important in many cellular and developmental processes and is relevant to human disease. Studies in D. melanogaster demonstrate that XRN1 can target specific RNAs, which have important consequences for developmental pathways. Osteosarcoma is a malignancy of the bone and accounts for 2% of all paediatric cancers worldwide. 5 year survival of patients has remained static since the 1970s and therefore furthering our molecular understanding of this disease is crucial. Previous work has shown a downregulation of XRN1 in osteosarcoma cells, however the transcripts regulated by XRN1 which might promote osteosarcoma remain elusive. Here, we confirm reduced levels of XRN1 in osteosarcoma cell lines and patient samples and identify XRN1-sensitive transcripts in human osteosarcoma cells. Using RNA-seq in XRN1-knockdown SAOS-2 cells, we show that 1178 genes are differentially regulated. Using a novel bioinformatic approach, we demonstrate that 134 transcripts show characteristics of direct post-transcriptional regulation by XRN1. Long non-coding RNAs (lncRNAs) are enriched in this group suggesting that XRN1 normally plays an important role in controlling lncRNA expression in these cells. Among potential lncRNAs targeted by XRN1 is HOTAIR, which is known to be upregulated in osteosarcoma and contribute to disease progression. We have also identified G-rich and GU motifs in post-transcriptionally regulated transcripts which appear to sensitise them to XRN1 degradation. Our results therefore provide significant insights into the specificity of XRN1 in human cells which is relevant to disease.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.08.10.244426v1?rss=1

Authors: Xue, B., Jordan, B., Rizvi, S., Naegle, K. M.

Abstract:

Tyrosine and serine/threonine kinases are essential regulators of cell processes and are important targets for human therapies. Unfortunately, very little is known about specific kinase-substrate relationships, making it difficult to infer meaning from dysregulated phosphoproteomic datasets or for researchers to identify possible kinases that regulate specific or novel phosphorylation sites. The last two decades have seen an explosion in algorithms to extrapolate from what little is known into the larger unknown -- predicting kinase-substrate relationships using a variety of approaches that include the sequence-specificity of kinase catalytic domains and various other factors, such as evolutionary relationships, co-expression, and protein-protein interaction networks. Unfortunately, a number of limitations prevent researchers from easily harnessing these resources, such as loss of resource accessibility, limited information in publishing that results in poor mapping to a human reference, using different kinase names, and not being updated to match the growth of the human phosphoproteome. Here, we propose a methodological framework for publishing predictions in a unified way, which entails ensuring predictions have been run on a current reference proteome, mapping the same substrates and kinases across resources to a common reference, filtering for the human phosphoproteome, and providing methods for updating the resource easily in the future. We applied this framework on three currently available resources, published in the last decade, which provide kinase-specific predictions in the human proteome. Using the unified datasets, we then explore the role of study bias, the emergent network properties of these predictors, and comparisons within and between predictors. The combination of the code for unification and analysis, as well as the unified predictors are available under the resource we named KinPred. We believe this resource will be useful for a wide range of applications and establishes best practices for future predictors for long-term usability and sustainability.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.08.10.244327v1?rss=1

Authors: Zinghirino, F., Pappalardo, X. G., Messina, A., Guarino, F., De Pinto, V.

Abstract:

Background: VDACs (Voltage Dependent Anion selective Channels) are pore forming proteins of the outer mitochondrial membrane, whose permeability is primarily due to their presence. In higher eukaryotes three isoforms raised during the evolution: they have the same exon-intron organization and the proteins show the same channel-forming activity. We provide a comprehensive analysis of the three human VDAC genes (VDAC1, VDAC2, VDAC3), their expression profiles, promoter activity, and potential transcriptional regulators. Results: VDAC isoforms are broadly but also specifically expressed in various human tissues at different levels with a predominance of VDAC1 and VDAC2 over VDAC3. However, RNA-seq CAGE approach revealed a higher level of transcription activation of VDAC3 gene. We experimentally confirmed this information by reporter assay of VDACs promoter activity. Transcription Factor Binding Sites (TFBSs) distribution in the promoters was investigated. The main regulators common to the three VDAC genes were identified as E2FF, NRF1, KLFS, EBOX transcription factors family members. All of them are involved in cell cycle and growth, proliferation, differentiation, apoptosis, and metabolism. More transcription factors specific for each isoform gene were identified, supporting the results in the literature, indicating a general role of VDAC1, as actor of apoptosis for VDAC2, and the involvement in sex determination and development of VDAC3. Conclusions: For the first time, we propose a comparative analysis of human VDAC promoters to investigate their specific biological functions. Bioinformatics and experimental results confirm the essential role of VDAC protein family in mitochondrial functionality. Moreover, insights about a specialized function and different regulation mechanisms arise for the three isoforms genes.

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