Virus-Induced Changes in mRNA Secondary Structure Uncover cis-Regulatory Elements that Directly Control Gene Expression.

mRNAs carry two layers of information, the genetic code and the information that dictates their post-transcriptional fate. The latter function relies on a complex interplay between cis-elements and trans-regulators, and unbiased identification of these elements is still challenging. To identify cis-elements that control gene expression, we use dimethyl sulfate (DMS) mutational profiling with sequencing and map changes in mRNA secondary structure following viral infection. Our dynamic structural data reveal a major role for ribosomes in unwinding secondary structures, which is further supported by the relationship we uncover between structure and translation efficiency. Moreover, our analysis revealed dozens of regions in viral and cellular mRNAs that exhibit changes in secondary structure. In-depth analysis of these regions reveals cis-elements in 3' UTRs that regulate mRNA stability and elements within coding sequences that control translation. Overall, our study demonstrates how mapping dynamic changes in mRNA structure allows unbiased identification of functional regulatory elements.

Shared sequence characteristics identified in non-canonical rearrangements of HSV-1 genomes.

IMPORTANCE: Mutations and genetic rearrangements are the primary driving forces of evolution. Viruses provide valuable model systems for investigating these mechanisms due to their rapid evolutionary rates and vast genetic variability. To investigate genetic rearrangements in the double-stranded DNA genome of herpes simplex virus type 1, the viral population was serially passaged in various cell types. The serial passaging led to formation of defective genomes, resulted from cell-specific non-canonical rearrangements (NCRs). Interestingly, we discovered shared sequence characteristics underlying the formation of these NCRs across all cell types. Moreover, most NCRs identified in clinical samples shared these characteristics. Based on our findings, we propose a model elucidating the formation of NCRs during viral replication within the nucleus of eukaryotic cells.

The Transcription and Translation Landscapes during Human Cytomegalovirus Infection Reveal Novel Host-Pathogen Interactions.

Viruses are by definition fully dependent on the cellular translation machinery, and develop diverse mechanisms to co-opt this machinery for their own benefit. Unlike many viruses, human cytomegalovirus (HCMV) does suppress the host translation machinery, and the extent to which translation machinery contributes to the overall pattern of viral replication and pathogenesis remains elusive. Here, we combine RNA sequencing and ribosomal profiling analyses to systematically address this question. By simultaneously examining the changes in transcription and translation along HCMV infection, we uncover extensive transcriptional control that dominates the response to infection, but also diverse and dynamic translational regulation for subsets of host genes. We were also able to show that, at late time points in infection, translation of viral mRNAs is higher than that of cellular mRNAs. Lastly, integration of our translation measurements with recent measurements of protein abundance enabled comprehensive identification of dozens of host proteins that are targeted for degradation during HCMV infection. Since targeted degradation indicates a strong biological importance, this approach should be applicable for discovering central host functions during viral infection. Our work provides a framework for studying the contribution of transcription, translation and degradation during infection with any virus.

1-(sulfonyl)-5-(arylsulfonyl)indoline as activators of the tumor cell specific M2 isoform of pyruvate kinase.

Cancer cells preferentially use glycolysis rather than oxidative phosphorylation for their rapid growth. They consume large amount of glucose to produce lactate even when oxygen is abundant, a phenomenon known as the Warburg effect. This metabolic change originates from a shift in the expression of alternative spliced isoforms of the glycolytic enzyme pyruvate kinase (PK), from PKM1 to PKM2. While PKM1 is constitutively active, PKM2 is switched from an inactive dimer form to an active tetramer form by small molecule activators. The prevalence of PKM2 in cancer cells relative to the prevalence of PKM1 in many normal cells, suggests a therapeutic strategy whereby activation of PKM2 may counter the abnormal cellular metabolism in cancer cells, and consequently decreased cellular proliferation. Herein we describe the discovery and optimization of a series of PKM2 activators derived from the 2-((2,3-dihydrobenzo[b][1,4] dioxin-6-yl)thio)-1-(2-methyl-1-(methylsulfonyl)indolin-5-yl) ethanone scaffold. The synthesis, SAR analysis, enzyme active site docking, enzymatic reaction kinetics, selectivity and pharmaceutical properties are discussed.

MuD: an interactive web server for the prediction of non-neutral substitutions using protein structural data.

The discrimination between functionally neutral amino acid substitutions and non-neutral mutations, affecting protein function, is very important for our understanding of diseases. The rapidly growing amounts of experimental data enable the development of computational tools to facilitate the annotation of these substitutions. Here, we describe a Random Forests-based classifier, named Mutation Detector (MuD) that utilizes structural and sequence-derived features to assess the impact of a given substitution on the protein function. In its automatic mode, MuD is comparable to alternative tools in performance. However, the uniqueness of MuD is that user-reported protein-specific structural and functional information can be added at run-time, thereby enhancing the prediction accuracy further. The MuD server, available at http://mud.tau.ac.il, assigns a reliability score to every prediction, thus offering a useful tool for the prioritization of substitutions in proteins with an available 3D structure.

A Myo6 mutation destroys coordination between the myosin heads, revealing new functions of myosin VI in the stereocilia of mammalian inner ear hair cells.

Myosin VI, found in organisms from Caenorhabditis elegans to humans, is essential for auditory and vestibular function in mammals, since genetic mutations lead to hearing impairment and vestibular dysfunction in both humans and mice. Here, we show that a missense mutation in this molecular motor in an ENU-generated mouse model, Tailchaser, disrupts myosin VI function. Structural changes in the Tailchaser hair bundles include mislocalization of the kinocilia and branching of stereocilia. Transfection of GFP-labeled myosin VI into epithelial cells and delivery of endocytic vesicles to the early endosome revealed that the mutant phenotype displays disrupted motor function. The actin-activated ATPase rates measured for the D179Y mutation are decreased, and indicate loss of coordination of the myosin VI heads or 'gating' in the dimer form. Proper coordination is required for walking processively along, or anchoring to, actin filaments, and is apparently destroyed by the proximity of the mutation to the nucleotide-binding pocket. This loss of myosin VI function may not allow myosin VI to transport its cargoes appropriately at the base and within the stereocilia, or to anchor the membrane of stereocilia to actin filaments via its cargos, both of which lead to structural changes in the stereocilia of myosin VI-impaired hair cells, and ultimately leading to deafness.

Small molecule correctors of F508del-CFTR discovered by structure-based virtual screening.

Folding correctors of F508del-CFTR were discovered by in silico structure-based screening utilizing homology models of CFTR. The intracellular segment of CFTR was modeled and three cavities were identified at inter-domain interfaces: (1) Interface between the two Nucleotide Binding Domains (NBDs); (2) Interface between NBD1 and Intracellular Loop (ICL) 4, in the region of the F508 deletion; (3) multi-domain interface between NBD1:2:ICL1:2:4. We hypothesized that compounds binding at these interfaces may improve the stability of the protein, potentially affecting the folding yield or surface stability. In silico structure-based screening was performed at the putative binding-sites and a total of 496 candidate compounds from all three sites were tested in functional assays. A total of 15 compounds, representing diverse chemotypes, were identified as F508del folding correctors. This corresponds to a 3% hit rate, ~tenfold higher than hit rates obtained in corresponding high-throughput screening campaigns. The same binding sites also yielded potentiators and, most notably, compounds with a dual corrector-potentiator activity (dual-acting). Compounds harboring both activity types may prove to be better leads for the development of CF therapeutics than either pure correctors or pure potentiators. To the best of our knowledge this is the first report of structure-based discovery of CFTR modulators.

Conserved interactions required for inhibition of the main protease of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

The COVID-19 pandemic caused by the SARS-CoV-2 requires a fast development of antiviral drugs. SARS-CoV-2 viral main protease (Mpro, also called 3C-like protease, 3CLpro) is a potential target for drug design. Crystal and co-crystal structures of the SARS-CoV-2 Mpro have been solved, enabling the rational design of inhibitory compounds. In this study we analyzed the available SARS-CoV-2 and the highly similar SARS-CoV-1 crystal structures. We identified within the active site of the Mpro, in addition to the inhibitory ligands' interaction with the catalytic C145, two key H-bond interactions with the conserved H163 and E166 residues. Both H-bond interactions are present in almost all co-crystals and are likely to occur also during the viral polypeptide cleavage process as suggested from docking of the Mpro cleavage recognition sequence. We screened in silico a library of 6900 FDA-approved drugs (ChEMBL) and filtered using these key interactions and selected 29 non-covalent compounds predicted to bind to the protease. Additional screen, using DOCKovalent was carried out on DrugBank library (11,414 experimental and approved drugs) and resulted in 6 covalent compounds. The selected compounds from both screens were tested in vitro by a protease activity inhibition assay. Two compounds showed activity at the 50 µM concentration range. Our analysis and findings can facilitate and focus the development of highly potent inhibitors against SARS-CoV-2 infection.

A systematic view on influenza induced host shutoff.

Host shutoff is a common strategy used by viruses to repress cellular mRNA translation and concomitantly allow the efficient translation of viral mRNAs. Here we use RNA-sequencing and ribosome profiling to explore the mechanisms that are being utilized by the Influenza A virus (IAV) to induce host shutoff. We show that viral transcripts are not preferentially translated and instead the decline in cellular protein synthesis is mediated by viral takeover on the mRNA pool. Our measurements also uncover strong variability in the levels of cellular transcripts reduction, revealing that short transcripts are less affected by IAV. Interestingly, these mRNAs that are refractory to IAV infection are enriched in cell maintenance processes such as oxidative phosphorylation. Furthermore, we show that the continuous oxidative phosphorylation activity is important for viral propagation. Our results advance our understanding of IAV-induced shutoff, and suggest a mechanism that facilitates the translation of genes with important housekeeping functions.

GeneAnalytics: An Integrative Gene Set Analysis Tool for Next Generation Sequencing, RNAseq and Microarray Data.

Postgenomics data are produced in large volumes by life sciences and clinical applications of novel omics diagnostics and therapeutics for precision medicine. To move from "data-to-knowledge-to-innovation," a crucial missing step in the current era is, however, our limited understanding of biological and clinical contexts associated with data. Prominent among the emerging remedies to this challenge are the gene set enrichment tools. This study reports on GeneAnalytics™ ( geneanalytics.genecards.org ), a comprehensive and easy-to-apply gene set analysis tool for rapid contextualization of expression patterns and functional signatures embedded in the postgenomics Big Data domains, such as Next Generation Sequencing (NGS), RNAseq, and microarray experiments. GeneAnalytics' differentiating features include in-depth evidence-based scoring algorithms, an intuitive user interface and proprietary unified data. GeneAnalytics employs the LifeMap Science's GeneCards suite, including the GeneCards®--the human gene database; the MalaCards-the human diseases database; and the PathCards--the biological pathways database. Expression-based analysis in GeneAnalytics relies on the LifeMap Discovery®--the embryonic development and stem cells database, which includes manually curated expression data for normal and diseased tissues, enabling advanced matching algorithm for gene-tissue association. This assists in evaluating differentiation protocols and discovering biomarkers for tissues and cells. Results are directly linked to gene, disease, or cell "cards" in the GeneCards suite. Future developments aim to enhance the GeneAnalytics algorithm as well as visualizations, employing varied graphical display items. Such attributes make GeneAnalytics a broadly applicable postgenomics data analyses and interpretation tool for translation of data to knowledge-based innovation in various Big Data fields such as precision medicine, ecogenomics, nutrigenomics, pharmacogenomics, vaccinomics, and others yet to emerge on the postgenomics horizon.

Human embryonic stem cell-derived neural crest cells capable of expressing markers of osteochondral or meningeal-choroid plexus differentiation.

AIMS: The transcriptome and fate potential of three diverse human embryonic stem cell-derived clonal embryonic progenitor cell lines with markers of cephalic neural crest are compared when differentiated in the presence of combinations of TGFß3, BMP4, SCF and HyStem-C matrices. MATERIALS & METHODS: The cell lines E69 and T42 were compared with MEL2, using gene expression microarrays, immunocytochemistry and ELISA. RESULTS: In the undifferentiated progenitor state, each line displayed unique markers of cranial neural crest including TFAP2A and CD24; however, none expressed distal HOX genes including HOXA2 or HOXB2, or the mesenchymal stem cell marker CD74. The lines also showed diverse responses when differentiated in the presence of exogenous BMP4, BMP4 and TGFß3, SCF, and SCF and TGFß3. The clones E69 and T42 showed a profound capacity for expression of endochondral ossification markers when differentiated in the presence of BMP4 and TGFß3, choroid plexus markers in the presence of BMP4 alone, and leptomeningeal markers when differentiated in SCF without TGFß3. CONCLUSION: The clones E69 and T42 may represent a scalable source of primitive cranial neural crest cells useful in the study of cranial embryology, and potentially cell-based therapy.

LifeMap Discovery™: the embryonic development, stem cells, and regenerative medicine research portal.

LifeMap Discovery™ provides investigators with an integrated database of embryonic development, stem cell biology and regenerative medicine. The hand-curated reconstruction of cell ontology with stem cell biology; including molecular, cellular, anatomical and disease-related information, provides efficient and easy-to-use, searchable research tools. The database collates in vivo and in vitro gene expression and guides translation from in vitro data to the clinical utility, and thus can be utilized as a powerful tool for research and discovery in stem cell biology, developmental biology, disease mechanisms and therapeutic discovery. LifeMap Discovery is freely available to academic nonprofit institutions at http://discovery.lifemapsc.com.