If someone else is doing the experiment, why should I do it? ...I have been strongly shaped by my personal interactions and training environments. Learning to work with flies was exhilarating, and ...the power of genetics seemed limitless. ...my proficiency in mammalian cell culture was a bonus at the time, as we began to set up high-throughput cell-based screening using the newly discovered RNAi system in fly cells. ...my love of screening has led me to start a Core at UPenn, which has led me to help the scientific community to apply this approach to diverse aspects of science but also led me to translational research, and we have begun screening acute leukemia patients for sensitivities to approved therapeutics to personalize therapies.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Picornaviruses are a widespread group of pathogens that can cause diverse pathologies. Pathogenesis is thought to be driven by the tissue-specific tropisms displayed by these viruses. For example, ...many picornaviruses can infect the heart and cause viral myocarditis. Encephalomyocarditis virus (EMCV) is a rodent pathogen that causes myocarditis in rodent models and has been used to model this biology. However, the receptor and entry requirements for this virus are poorly understood. L. E. Bazzone, M. King, C. R. MacKay, P. P. Kyawe, et al. (mBio 10:e02734-18, 2019, https://doi.org/10.1128/mBio.02734-18) tackled this problem using CRISPR knockout screening in human cells that are susceptible to EMCV and identified ADAM9 as an essential entry factor for EMCV in mouse and human cells. Since the extracellular domain but not the enzymatic activity or intracellular domain is required for infection, the data suggest that ADAM9 acts as an entry receptor or at an early step in the process, shedding light on the biology of EMCV infection and pathogenesis.
Over the past decade there has been increased awareness of the potential role of alternative splicing in the etiology of cancer. In particular, advances in RNA-Sequencing technology and analysis has ...led to a wave of discoveries in the last few years regarding the causes and functional relevance of alternative splicing in cancer. Here we discuss the current understanding of the connections between splicing and cancer, with a focus on the most recent findings. We also discuss remaining questions and challenges that must be addressed in order to use our knowledge of splicing to guide the diagnosis and treatment of cancer.
Flaviviruses enter host cells through the process of clathrin-mediated endocytosis, and the spectrum of host factors required for this process are incompletely understood. Here we found that ...lymphocyte antigen 6 locus E (LY6E) promotes the internalization of multiple flaviviruses, including West Nile virus, Zika virus, and dengue virus. Perhaps surprisingly, LY6E is dispensable for the internalization of the endogenous cargo transferrin, which is also dependent on clathrin-mediated endocytosis for uptake. Since viruses are substantially larger than transferrin, we reasoned that LY6E may be required for uptake of larger cargoes and tested this using transferrin-coated beads of similar size as flaviviruses. LY6E was indeed required for the internalization of transferrin-coated beads, suggesting that LY6E is selectively required for large cargo. Cell biological studies found that LY6E forms tubules upon viral infection and bead internalization, and we found that tubule formation was dependent on RNASEK, which is also required for flavivirus internalization, but not transferrin uptake. Indeed, we found that RNASEK is also required for the internalization of transferrin-coated beads, suggesting it functions upstream of LY6E. These LY6E tubules resembled microtubules, and we found that microtubule assembly was required for their formation and flavivirus uptake. Since microtubule end-binding proteins link microtubules to downstream activities, we screened the three end-binding proteins and found that EB3 promotes virus uptake and LY6E tubularization. Taken together, these results highlight a specialized pathway required for the uptake of large clathrin-dependent endocytosis cargoes, including flaviviruses.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
The recent outbreak of Zika virus (ZIKV) infection in the Americas and its devastating impact on fetal development have prompted the World Health Organization (WHO) to declare the ZIKV pandemic as a ...Public Health Emergency of International Concern. Rapid and reliable diagnostics for ZIKV are vital because ZIKV-infected individuals display no symptoms or nonspecific symptoms similar to other viral infections. Because immunoassays lack adequate sensitivity and selectivity and are unable to identify active state of infection, molecular diagnostics are an effective means to detect ZIKV soon after infection and throughout pregnancy. We report on a highly sensitive reverse-transcription loop-mediated, isothermal amplification (RT-LAMP) assay for rapid detection of ZIKV and its implementation in a simple, easy-to-use, inexpensive, point-of-care (POC) disposable cassette that carries out all the unit operations from sample introduction to detection. For thermal control of the cassette, we use a chemically heated cup without a need for electrical power. Amplification products are detected with leuco crystal violet (LCV) dye by eye without a need for instrumentation. We demonstrated the utility of our POC diagnostic system by detecting ZIKV in oral samples with sensitivity of 5 plaque-forming units (PFU) in less than 40 min. Our system is particularly suitable for resource-poor settings, where centralized laboratory facilities, funds, and trained personnel are in short supply, and for use in doctors’ offices, clinics, and at home.
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IJS, KILJ, NUK, PNG, UL, UM
Many eukaryotic genes generate linear mRNAs and circular RNAs, but it is largely unknown how the ratio of linear to circular RNA is controlled or modulated. Using RNAi screening in Drosophila cells, ...we identify many core spliceosome and transcription termination factors that control the RNA outputs of reporter and endogenous genes. When spliceosome components were depleted or inhibited pharmacologically, the steady-state levels of circular RNAs increased while expression of their associated linear mRNAs concomitantly decreased. Upon inhibiting RNA polymerase II termination via depletion of the cleavage/polyadenylation machinery, circular RNA levels were similarly increased. This is because readthrough transcripts now extend into downstream genes and are subjected to backsplicing. In total, these results demonstrate that inhibition or slowing of canonical pre-mRNA processing events shifts the steady-state output of protein-coding genes toward circular RNAs. This is in part because nascent RNAs become directed into alternative pathways that lead to circular RNA production.
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•RNAi screen identifies pre-mRNA processing factors that modulate circular RNA levels•Circular RNA expression increases when core spliceosomal components are depleted•Backsplicing can occur in conditions that inhibit canonical mRNA splicing events•Readthrough transcription enables production of circular RNAs from downstream genes
Many protein-coding genes produce linear mRNAs and circular RNAs. Liang et al. find that circular RNAs become the preferred gene output when core spliceosome or transcription termination factors are depleted from cells. This is in part because nascent RNAs are directed into alternative pathways that lead to circular RNA biogenesis.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The détente between pathogen and host has been of keen interest to researchers in spite of being exceedingly difficult to probe. Recently, new RNA interference (RNAi) technologies, in particular in ...Drosophila tissue culture cells, have made it possible to interrogate the genetics of host organisms rapidly, with nearly complete genomic coverage and high fidelity. Therefore, it is not surprising that the applications of RNAi to the study of host–pathogen interactions were among the first to be published and have already revealed many new insights into the hosts’ role in infection. This review will highlight the application of RNAi screening to pathogen–host interactions in Drosophila cells and will reveal some of the lessons learned from this approach.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Intrinsic innate immune mechanisms are the first line of defense against pathogens and exist to control infection autonomously in infected cells. Here, we showed that autophagy, an intrinsic ...mechanism that can degrade cytoplasmic components, played a direct antiviral role against the mammalian viral pathogen vesicular stomatitis virus (VSV) in the model organism
Drosophila. We found that the surface glycoprotein, VSV-G, was likely the pathogen-associated molecular pattern (PAMP) that initiated this cell-autonomous response. Once activated, autophagy decreased viral replication, and repression of autophagy led to increased viral replication and pathogenesis in cells and animals. Lastly, we showed that the antiviral response was controlled by the phosphatidylinositol 3-kinase (PI3K)-Akt-signaling pathway, which normally regulates autophagy in response to nutrient availability. Altogether, these data uncover an intrinsic antiviral program that links viral recognition to the evolutionarily conserved nutrient-signaling and autophagy pathways.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Thousands of eukaryotic protein-coding genes are noncanonically spliced to produce circular RNAs. Bioinformatics has indicated that long introns generally flank exons that circularize in Drosophila, ...but the underlying mechanisms by which these circular RNAs are generated are largely unknown. Here, using extensive mutagenesis of expression plasmids and RNAi screening, we reveal that circularization of the Drosophila laccase2 gene is regulated by both intronic repeats and trans-acting splicing factors. Analogous to what has been observed in humans and mice, base-pairing between highly complementary transposable elements facilitates backsplicing. Long flanking repeats (∼ 400 nucleotides nt) promote circularization cotranscriptionally, whereas pre-mRNAs containing minimal repeats (<40 nt) generate circular RNAs predominately after 3' end processing. Unlike the previously characterized Muscleblind (Mbl) circular RNA, which requires the Mbl protein for its biogenesis, we found that Laccase2 circular RNA levels are not controlled by Mbl or the Laccase2 gene product but rather by multiple hnRNP (heterogeneous nuclear ribonucleoprotein) and SR (serine-arginine) proteins acting in a combinatorial manner. hnRNP and SR proteins also regulate the expression of other Drosophila circular RNAs, including Plexin A (PlexA), suggesting a common strategy for regulating backsplicing. Furthermore, the laccase2 flanking introns support efficient circularization of diverse exons in Drosophila and human cells, providing a new tool for exploring the functional consequences of circular RNA expression across eukaryotes.
The main protease (Mpro) of SARS-CoV-2 is a validated antiviral drug target. Several Mpro inhibitors have been reported with potent enzymatic inhibition and cellular antiviral activity, including ...GC376, boceprevir, calpain inhibitors II, and XII, with each containing a reactive warhead that covalently modifies the catalytic Cys145. Coupling structure-based drug design with the one-pot Ugi four-component reaction, we discovered one of the most potent noncovalent inhibitors, 23R (Jun8-76-3A) that is structurally distinct from the canonical Mpro inhibitor GC376. Significantly, 23R is highly selective compared with covalent inhibitors such as GC376, especially toward host proteases. The cocrystal structure of SARS-CoV-2 Mpro with 23R revealed a previously unexplored binding site located in between the S2 and S4 pockets. Overall, this study discovered 23R, one of the most potent and selective noncovalent SARS-CoV-2 Mpro inhibitors reported to date, and a novel binding pocket in Mpro that can be explored for inhibitor design.
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