PaperPlayer biorxiv molecular biology

Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.08.03.234013v1?rss=1

Authors: Cao, H., Zhang, X., Ruan, Y., Zhang, L., Cui, Z., Li, X., Jia, B.

Abstract:

Callus formation and adventitious shoot differentiation could be observed on the cut surface of completely decapitated tomato plants. We propose that this process can be used as a model system to investigate the mechanisms that regulate indirect regeneration of higher plants without the addition of exogenous hormones. This study analyzed the patterns of trans-zeatin and miRNA expression during in vivo regeneration of tomato. Analysis of trans-zeatin revealed that the hormone cytokinin played an important role in in vivo regeneration of tomato. Among 183 miRNAs and 1168 predicted target genes sequences identified, 93 miRNAs and 505 potential targets were selected based on differential expression levels for further characterization. Expression patterns of six miRNAs, including sly-miR166, sly-miR167, sly-miR396, sly-miR397, novel 156, and novel 128, were further validated by qRT-PCR. We speculate that sly-miR156, sly-miR160, sly-miR166, and sly-miR397 play major roles in callus formation of tomato during in vivo regeneration by regulating cytokinin, IAA, and laccase levels. Overall, our microRNA sequence and target analyses of callus formation during in vivo regeneration of tomato provide novel insights into the regulation of regeneration in higher plants.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.08.03.235143v1?rss=1

Authors: Chaves, D. A., Hui, D., Li, L., Moresco, J. J., Oh, M. E., Conte, D., Yates, J. R., Mello, C. C., Gu, W.

Abstract:

Eukaryotic cells regulate 5' triphosphorylated (ppp-) RNAs to promote cellular functions and prevent recognition by antiviral RNA sensors. For example, RNA capping enzymes possess triphosphatase domains that remove the {gamma} phosphates of ppp-RNAs during RNA capping. Members of the closely related PIR1 family of RNA polyphosphatases remove both the {beta} and {gamma} phosphates from ppp-RNAs. Here we show that C. elegans PIR-1 dephosphorylates ppp-RNAs made by cellular RdRPs and is required for the maturation of 26G-RNAs, Dicer-dependent small RNAs that regulate thousands of genes during spermatogenesis and embryogenesis. PIR-1 also regulates the CSR-1 22G-RNA pathway and has critical functions in both somatic and germline development. Our findings suggest that PIR-1 modulates both Dicer-dependent and -independent Argonaute pathways, and provide insight into how cells and viruses use a conserved RNA phosphatase to regulate and respond to ppp-RNA species.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.08.04.234948v1?rss=1

Authors: Sendino, M., Omaetxebarria, M. J., Prieto, G., Rodriguez, J. A.

Abstract:

The nuclear export receptor CRM1 (XPO1) recognizes and binds specific sequence motifs termed nuclear export signals (NESs) in cargo proteins. About 200 NES motifs have been identified, but over a thousand human proteins are potential CRM1 cargos, and most of their NESs remain to be identified. On the other hand, the interaction of NES peptides with the 'NES-binding groove' of CRM1 has been studied in detail using structural and biochemical analyses, but a better understanding of CRM1 function requires further investigation of how the results from these in vitro studies translate into actual NES export in a cellular context. Here we show that a simple cellular assay, based on a recently described reporter (SRVB/A), can be applied to identify novel potential NESs motifs, and to obtain relevant information on different aspects of CRM1-mediated NES export. Using cellular assays, we first map 19 new sequence motifs with nuclear export activity in 14 cancer-related proteins that are potential CRM1 cargos. Next, we investigate the effect of mutations in individual NES-binding groove residues, providing further insight into CRM1-mediated NES export. Finally, we extend the search for CRM1-dependent NESs to a recently uncovered, but potentially vast, set of small proteins called micropeptides. By doing so, we report the first NES-harbouring human micropeptides.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.08.04.234880v1?rss=1

Authors: Speranskaya, A., Kaptelova, V., Valdokhina, A., Bulanenko, V., Samoilov, A., Korneenko, E., Shipulina, O., Akimkin, V.

Abstract:

Here we provide technical data for amplifying the complete genome of SARS-CoV-2 from clinical samples using only seventeen pairs of primers. We demonstrate that the CV2000bp primer panel successfully produces genomes when used with the residual total RNA extracts from positive clinical samples following diagnostic RT-PCRs (with Ct in the range from 13 to 20). The library preparation method reported here includes genome amplification of ~1750-2000 bp fragments followed by ultrasonic fragmentation combined with the introduction of Illumina compatible adapters. Using the SCV2000bp panel, 25 complete SARS-CoV-2 virus genome sequences were sequenced from clinical samples of COVID-19 patients from Moscow obtained in late March - early April.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.08.04.200550v1?rss=1

Authors: Pokrovsky, D., Forne, I., Straub, T., Imhof, A., Rupp, R.

Abstract:

Forming an embryo from a zygote poses an apparent conflict for epigenetic regulation. On one hand, the de novo induction of cell fate identities requires the establishment and subsequent maintenance of epigenetic information to harnish developmental gene expression. On the other hand, the embryo depends on cell proliferation, and every round of DNA replication dilutes preexisting histone modifications by incorporation of new unmodified histones into chromatin. Here we investigated the possible relationship between the propagation of epigenetic information and the developmental cell proliferation during Xenopus embryogenesis. We systemically inhibited cell proliferation during the G1/S-transition in gastrula embryos and followed their development until the tadpole stage. Comparing wild-type and cell cycle-arrested embryos, we show that the inhibition of cell proliferation is principally compatible with embryo survival and cellular differentiation. In parallel, we quantified by mass spectrometry the abundance of a large set of histone modification states, which reflects the developmental maturation of the embryonic epigenome. The arrested embryos developed abnormal stage-specific histone modification profiles, in which transcriptionally repressive histone marks were overrepresented. Embryos released from the cell cycle block during neurulation reverted back towards normality on morphological, molecular and epigenetic levels. These results indicate that replicational dilution of histone marks has a strong impact on developmental chromatin maturation. We propose that this influence is strong enough to control developmental decisions, specifically in cell populations that switch between resting and proliferating states such as stem cells.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.08.03.235572v1?rss=1

Authors: Urbanek, K., Sutherland, D. M., Orchard, R. C., Wilen, C. B., Knowlton, J. J., Aravamudhan, P., Taylor, G. M., Virgin, H. W., Dermody, T. S.

Abstract:

Engagement of cell-surface receptors by viruses is a critical determinant of viral tropism and disease. The reovirus attachment protein, {sigma}1, binds sialylated glycans and proteinaceous receptors to mediate infection, but the specific requirements on different cell types are unknown. To identify host factors required for reovirus-induced cell death, we conducted a CRISPR-knockout screen targeting over 20,000 genes in murine microglial BV2 cells. Candidate genes identified as required for reovirus to cause cell death were highly enriched for sialic acid synthesis and transport. Two of the top candidates identified, cytidine monophosphate N-acetylneuraminic acid synthetase (Cmas) and solute carrier family 35 member A1 (Slc35a1), promote sialic acid expression on the cell surface. Two reovirus strains differing in the capacity to bind sialic acid, T3SA+ and T3SA-, were used to evaluate Cmas and Slc35a1 as potential host genes required for infection. Following CRISPR-Cas9 disruption of either gene, cell-surface expression of sialic acid was diminished. These results correlated with decreased binding of strain T3SA+, which is capable of engaging sialic acid. Disruption of either gene did not alter the low-level binding of T3SA-, which does not engage sialic acid. Infectivity of T3SA+ was diminished to levels of T3SA- in cells lacking Cmas and Slc35a1 by CRISPR ablation. However, exogenous expression of Cmas and Slc35a1 into the respective null cells restored sialic acid expression and T3SA+ binding and infectivity. These results demonstrate that Cmas and Slc35a1, which mediate cell-surface expression of sialic acid, are required in murine microglial cells for efficient reovirus binding and infection.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.08.03.233924v1?rss=1

Authors: Dobreva, M. P., Lynton-Jenkins, J. G., Chaves, J. A., Tokita, M., Bonneaud, C., Abzhanov, A.

Abstract:

Darwins finches, endemic to the Galapagos and Cocos islands, are an iconic example of adaptive radiation and evolution under natural selection. Comparative genetic studies using embryos of Darwins finches have shed light on the possible evolutionary processes underlying the speciation of this clade. Molecular identification of the sex of embryonic samples is important for such studies, where this information often cannot be inferred otherwise. We tested a fast and simple chicken embryo protocol for extraction of genomic DNA on Darwins finch embryos. In addition, we suggest modifications to two of the previously reported PCR primer sets for CHD1 , a gene used for sexing in adult passerine birds. The sex of all 29 tested embryos of six species of Darwins finches was determined successfully by PCR, using both primer sets. Hatchlings/nestlings and fledglings are also impossible to distinguish visually. This includes juveniles of sexually dimorphic species which are yet to moult in adult-like plumage and beak colouration. Furthermore, four species of Darwins finches are monomorphic, males and females looking alike. Therefore, sex assessment in the field can be a source of error, especially with respect to juveniles and mature monomorphic birds outside of the mating season. We caught 567 juveniles and adults belonging to six species of Darwins finches and only 44% had unambiguous sex-specific morphology. We sexed 363 birds by PCR, including individuals sexed based on marginal sex specific morphological traits (N=278) and birds which were impossible to classify in the field (N=39). For birds with marginal sex specific traits, PCR results revealed a 13% sexing error rate. This demonstrates that PCR based sexing can improve field studies on Darwins finches, especially when individuals with unclear sex-related morphology are involved. The protocols used here provide an easy and reliable way to sex Darwins finches throughout ontogeny, from embryos to adults.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.08.03.233791v1?rss=1

Authors: Whitworth, L., Coxon, J., van Laarhoven, A., Thuong, N. T. T., Dian, S., Alisjahbana, B., Ganiem, A. R., van Crevel, R., Thwaites, G., Troll, M., Edelstein, P., Sewell, R., Ramakrishnan, L.

Abstract:

Tuberculous meningitis (TBM) remains the most devastating form of tuberculosis (TB) with high mortality despite effective antimicrobial treatment. As mortality has been linked to excessive inflammation, anti-inflammatory glucocorticoids are now routinely used as adjunctive treatment with antimicrobial therapy. However, they reduce mortality by only ~ 30%, raising the possibility that only a subset of TBM deaths are caused by inflammatory pathophysiology. Studies in Vietnam found that the survival benefit of adjunctive glucocorticoids was limited to individuals with a common promoter variant in the leukotriene A4 hydrolase (LTA4H) gene encoding an enzyme that regulates inflammatory eicosanoid expression. The variant constitutes a C/T transition with TT homozygotes having increased expression over CT heterozygotes and CC homozygotes. In Vietnam, the LTA4H TT genotype predicted survival, consistent with dexamethasone benefiting only those individuals with a dysregulated hyper-inflammatory response. However, a study of TBM patients in Indonesia did not find the LTA4H TT genotype to confer a significant survival benefit. Given the potential of personalized life-saving antiinflammatory therapies guided by LTA4H genotype, we have used Bayesian methods to analyze the data from both studies. Bayesian analysis reveals that the LTA4H TT genotype confers survival benefit in both the Vietnam and Indonesia cohorts that begins within days and continues long-term. However, its benefit is nullified in the most severe cases where other factors cause early mortality. LTA4H TT genotype is associated with increased survival in HIV-positive patients also. Thus, our analysis extends the association of LTA4H genotype with TBM survival to populations outside of Vietnam and to HIV-positive patients. Patient LTA4H genotyping used in conjunction with disease severity assessment may help to target glucocorticoids to patients most likely to benefit from this broadly-acting immunosuppressive regimen despite its significant adverse effects.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.08.03.233957v1?rss=1

Authors: Bermudez-Guzman, L., Jimenez-Huezo, G., Arguedas, A., Leal, A.

Abstract:

The molecular function of a protein relies on its structure. Understanding how mutations alter structure and function in multi-domain proteins, is key to elucidate how a pathological phenotype is generated. However, one may fall into the logical bias of assessing protein damage only based on the mutations that are viable (survivorship bias), which can lead to partial conclusions. This is the case of PNKP, an important nuclear and mitochondrial DNA repair enzyme with kinase and phosphatase function. Most mutations in PNKP are confined to the kinase domain, leading to a pathological spectrum of three apparently distinct clinical entities. Since proteins and domains may have a different tolerance to disease causing mutations, we evaluated whether mutations in PNKP are under survivorship bias. Even when all mutations in the kinase domain are deleterious, we found a mayor mutation tolerability landscape in terms of survival. Instead, the phosphatase domain is less tolerant due to its low mutation rates, higher degree of sequence conservation, lower dN/dS ratios, and more disease-propensity hotspots. Thus, in multi-domain proteins, we propose the term "Wald's domain" for those who are not apparently more associated with disease, but that are less resistant to mutations in terms of survival. Together, our results support previous experimental evidence that demonstrated that the phosphatase domain is functionally more necessary and relevant for DNA repair, especially in the context of the development of the central nervous system. Thus, this bias should be taken into account when analyzing the mutational landscape in protein structure, function, and finally in disease.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.07.31.231555v1?rss=1

Authors: Ummarino, S., Bassal, M. A., Zhang, Y., Seelam, A. J., Kobayashi, I. S., Borchiellini, M., Ebralidze, A. K., Trinh, B. Q., Kobayashi, S. S., Di Ruscio, A.

Abstract:

Nutritional intake impacts the human epigenome by directing epigenetic pathways in normal cell development via as yet unknown molecular mechanisms. Consequently, imbalance in the nutritional intake is able to dysregulate the epigenetic profile and drive cells towards malignant transformation. Herein, we present a novel epigenetic effect of the essential nutrient, NAD. We demonstrate that impairment of DNMT1 enzymatic activity by NAD-promoted ADP-ribosylation, leads to demethylation and transcriptional activation of CEBPA gene, suggesting the existence of an unknown NAD-controlled region within the locus. In addition to the molecular events, NAD treated cells exhibit significant morphological and phenotypical changes that correspond to myeloid differentiation. Collectively, these results delineate a novel role for NAD in cell differentiation and indicate novel nutri-epigenetic strategy to regulate and control gene expression in human cells.

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