Biological insertion of computationally designed short transmembrane segments

The great majority of helical membrane proteins are inserted co-translationally into the ER membrane through a continuous ribosome-translocon channel. The efficiency of membrane insertion depends on transmembrane (TM) helix amino acid composition, the helix length and the position of the amino acids within the helix. In this work, we conducted a computational analysis of the composition and location of amino acids in transmembrane helices found in membrane proteins of known structure to obtain an extensive set of designed polypeptide segments with naturally occurring amino acid distributions. Then, using an in vitro translation system in the presence of biological membranes, we experimentally validated our predictions by analyzing its membrane integration capacity. Coupled with known strategies to control membrane protein topology, these findings may pave the way to de novo membrane protein design

Principles of 3D chromosome folding and evolutionary genome reshuffling in mammals.

Studying the similarities and differences in genomic interactions between species provides fertile grounds for determining the evolutionary dynamics underpinning genome function and speciation. Here, we describe the principles of 3D genome folding in vertebrates and show how lineage-specific patterns of genome reshuffling can result in different chromatin configurations. We (1) identified different patterns of chromosome folding in across vertebrate species (centromere clustering versus chromosomal territories); (2) reconstructed ancestral marsupial and afrotherian genomes analyzing whole-genome sequences of species representative of the major therian phylogroups; (3) detected lineage-specific chromosome rearrangements; and (4) identified the dynamics of the structural properties of genome reshuffling through therian evolution. We present evidence of chromatin configurational changes that result from ancestral inversions and fusions/fissions. We catalog the close interplay between chromatin higher-order organization and therian genome evolution and introduce an interpretative hypothesis that explains how chromatin folding influences evolutionary patterns of genome reshuffling. [Abstract copyright: Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.

Software for predicting the 3D structure of RNA molecules

RNA is not regarded anymore as a simple transfer molecule between DNA and proteins. Indeed, over the past decades a plethora of new functional roles have been assigned to RNA molecules. Such functions are carried out either by RNA molecules alone or through interactions with DNA, other RNA molecules, or proteins. In all cases, the structure that the RNA molecule adopts will impact its function, as it happens with proteins. Therefore, to fully characterize the function of an RNA molecule, its structure needs to be either determined by experiments or predicted by computation. Unfortunately, our knowledge of the atomic mechanism by which RNA molecules adopt their biological active structures is still limited. Such hurdle is now being addressed by the development of new computational methods for RNA structure prediction, which complement experimental methods such as X-ray crystallography, nuclear magnetic resonance, small-angle X-ray scattering, and cryo-electron microscopy. This software focus is not dedicated to a single computational method but aims at outlining the most adopted methods for computational RNA structure prediction

Communicating genome architecture: biovisualization of the genome, from data analysis and hypothesis generation to communication and learning

Genome discoveries at the core of biology are made by visual description and exploration of the cell, from microscopic sketches and biochemical mapping to computational analysis and spatial modeling. We outline the experimental and visualization techniques that have been developed recently which capture the three-dimensional interactions regulating how genes are expressed. We detail the challenges faced in integration of the data to portray the components and organization and their dynamic landscape. The goal is more than a single data-driven representation as interactive visualization for de novo research is paramount to decipher insights on genome organization in space.We received funding from the European Research Council under the European Union's Seventh Framework Programme (FP7/2007-2013)/ERC Synergy under grant agreement no 609989 (4Dgenome) as well as under the European Union's Horizon 2020 research and innovation program (grant agreement 676556). We also acknowledge the co-financing by the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) with funds from the European Regional Development Fund (ERDF) corresponding to the 2014-2020 Smart Growth Operating Program, the support of the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) to the EMBL partnership (BFU2017-85926-P) and through the Instituto de Salud Carlos III, the Centro de Excelencia Severo Ochoa (SEV-2012-0208), the CERCA Programme / Generalitat de Catalunya, and the Generalitat de Catalunya through Departament de Salut and Departament d'Empresa i Coneixement. This work reflects only the authors' views and the funding bodies are not liable for any use that may be made of the information contained therein

The anti-immune dengue subgenomic flaviviral RNA is present in vesicles in mosquito saliva and is associated with increased infectivity

Mosquito transmission of dengue viruses to humans starts with infection of skin resident cells at the biting site. There is great interest in identifying transmission-enhancing factors in mosquito saliva in order to counteract them. Here we report the discovery of high levels of the anti-immune subgenomic flaviviral RNA (sfRNA) in dengue virus 2-infected mosquito saliva. We established that sfRNA is present in saliva using three different methods: northern blot, RT-qPCR and RNA sequencing. We next show that salivary sfRNA is protected in detergent-sensitive compartments, likely extracellular vesicles. In support of this hypothesis, we visualized viral RNAs in vesicles in mosquito saliva and noted a marked enrichment of signal from 3'UTR sequences, which is consistent with the presence of sfRNA. Furthermore, we show that incubation with mosquito saliva containing higher sfRNA levels results in higher virus infectivity in a human hepatoma cell line and human primary dermal fibroblasts. Transfection of 3'UTR RNA prior to DENV2 infection inhibited type I and III interferon induction and signaling, and enhanced viral replication. Therefore, we posit that sfRNA present in salivary extracellular vesicles is delivered to cells at the biting site to inhibit innate immunity and enhance dengue virus transmission.Support for this research came from a fellowship from the McLauglin Family Foundation to TS, scholarships from the Fondation pour la Recherche Médicale (FRM project SPF202110013925) to HM, from the Institut Méditerranéen Hospitalier (IHU, Marseille) to IMSP and from the graduate school French Ministry of Higher Education and Research to FRC and FR, UTMB start-up funds and Cancer Prevention & Research Institute of Texas grant RP200650 to LJK and MKS, the Ministerio de Ciencia e Innovación (PID2020-115696RB-I00) to MAM-R, Cancer Prevention & Research Institute of Texas grant ID RR210018 to GN, Ministry of Education (Singapore) Tier3 grant (MOE2015-T3-1-003) to RMK and JP, a National Medical Research Council (Singapore) ZRRF grant (ZRRF/007/2017) to JP, a French Agence Nationale de la Recherche grant (ANR-20-CE15-0006) to JP, and the Duke-NUS Signature Research Programme funded by the Agency for Science Technology and Research (A*Star Singapore) and NIH/NIAID P01 AI150585 to MAGB. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Rational design of non-resistant targeted cancer therapies

Drug resistance is one of the major problems in targeted cancer therapy. A major cause of resistance is changes in the amino acids that form the drug-target binding site. Despite of the numerous efforts made to individually understand and overcome these mutations, there is a lack of comprehensive analysis of the mutational landscape that can prospectively estimate drug-resistance mutations. Here we describe and computationally validate a framework that combines the cancer-specific likelihood with the resistance impact to enable the detection of single point mutations with the highest chance to be responsible of resistance to a particular targeted cancer therapy. Moreover, for these treatment-threatening mutations, the model proposes alternative therapies overcoming the resistance. We exemplified the applicability of the model using EGFR-gefitinib treatment for Lung Adenocarcinoma (LUAD) and Lung Squamous Cell Cancer (LSCC) and the ERK2-VTX11e treatment for melanoma and colorectal cancer. Our model correctly identified the phenotype known resistance mutations, including the classic EGFR-T790M and the ERK2-P58L/S/T mutations. Moreover, the model predicted new previously undescribed mutations as potentially responsible of drug resistance. Finally, we provided a map of the predicted sensitivity of alternative ERK2 and EGFR inhibitors, with a particular highlight of two molecules with a low predicted resistance impact.The project was supported by the Spanish MINECO to M.A.M.-R. (BFU2010-19310). We also acknowledge the support of the Spanish Ministry of Economy and Competitiveness, Centro de Excelencia Severo Ochoa 2013-2017 (SEV-2012-0208) and the CERCA Programme of the Generalitat de Catalunya

Walking along chromosomes with super-resolution imaging, contact maps, and integrative modeling

Chromosome organization is crucial for genome function. Here, we present a method for visualizing chromosomal DNA at super-resolution and then integrating Hi-C data to produce three-dimensional models of chromosome organization. Using the super-resolution microscopy methods of OligoSTORM and OligoDNA-PAINT, we trace 8 megabases of human chromosome 19, visualizing structures ranging in size from a few kilobases to over a megabase. Focusing on chromosomal regions that contribute to compartments, we discover distinct structures that, in spite of considerable variability, can predict whether such regions correspond to active (A-type) or inactive (B-type) compartments. Imaging through the depths of entire nuclei, we capture pairs of homologous regions in diploid cells, obtaining evidence that maternal and paternal homologous regions can be differentially organized. Finally, using restraint-based modeling to integrate imaging and Hi-C data, we implement a method-integrative modeling of genomic regions (IMGR)-to increase the genomic resolution of our traces to 10 kb.This work was supported by funds from Ministerio de Ciencia, Innovación y Universidades of Spain (http://www.ciencia.gob.es/) (IJCI-2015-23352) to IF, Damon Runyon Cancer Research Foundation (https://www.damonrunyon.org/) and Howard Hughes Medical Institute (https://www.hhmi.org/) to BJB, Uehara Memorial Foundation Research (https://www.taisho-holdings.co.jp/en/environment/social/sciences/) to HMS, William Randolph Hearst Foundation (https://www.hearstfdn.org/) to RBM, EMBO (Long-Term fellowship) (https://www.embo.org/) to JE, NSF (Center for Theoretical Biological Physics, Rice University) (https://www.nsf.gov/) to MDP and JNO, NSF (CCF-1054898, CCF-1317291) (https://www.nsf.gov/), NIH (1R01EB018659-01, 1-U01- MH106011-01) (https://www.nih.gov/), and Office of Naval Research (N00014-13-1-0593, N00014-14-1-0610, N00014-16-1-2182, N00014-16-1- 2410) (https://www.onr.navy.mil/) to PY, NIH (1DP2OD008540, U01HL130010, UM1HG009375, 4DP2OD008540) (https://www.nih.gov/), NSF (PHY-1427654) (https://www.nsf.gov/), USDA (2017-05741) (https://www.usda.gov/), Welch Foundation (Q-1866) (http://www.welch1.org/), NVIDIA (https://www.nvidia.com/en-us/), IBM (https://www.ibm.com/us-en/?lnk=m), Google (https://www.google.com/), Cancer Prevention Research Institute of Texas (R1304) (http://www.cprit.state.tx.us/), and McNair Medical Institute (http://www.mcnairfoundation.org/what-we-fund/mcnair-medical-institute/) to E.L.A., Horizon 2020 Research and Innovation Programme (676556) (https://ec.europa.eu/programmes/horizon2020/en/), European Research Council (609989) (https://erc.europa.eu/), Ministerio de Ciencia, Innovación y Universidades of Spain (BFU2017-85926-P) (http://www.ciencia.gob.es/), CERCA, and AGAUR Programme of the Generalitat de Catalunya and Centros de Excelencia Severo Ochoa (SEV-2012-0208) (http://www.ciencia.gob.es/portal/site/MICINN/menuitem.7eeac5cd345b4f34f09dfd1001432ea0/?vgnextoid=cba733a6368c2310VgnVCM1000001d04140aRCRD) to M.A.M-R., and NIH (5DP1GM106412, R01HD091797, R01GM123289) (https://www.nih.gov/) to C-tW. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Hierarchical chromatin organization detected by TADpole

The rapid development of Chromosome Conformation Capture (3C-based techniques), as well as imaging together with bioinformatics analyses, has been fundamental for unveiling that chromosomes are organized into the so-called topologically associating domains or TADs. While TADs appear as nested patterns in the 3C-based interaction matrices, the vast majority of available TAD callers are based on the hypothesis that TADs are individual and unrelated chromatin structures. Here we introduce TADpole, a computational tool designed to identify and analyze the entire hierarchy of TADs in intra-chromosomal interaction matrices. TADpole combines principal component analysis and constrained hierarchical clustering to provide a set of significant hierarchical chromatin levels in a genomic region of interest. TADpole is robust to data resolution, normalization strategy and sequencing depth. Domain borders defined by TADpole are enriched in main architectural proteins (CTCF and cohesin complex subunits) and in the histone mark H3K4me3, while their domain bodies, depending on their activation-state, are enriched in either H3K36me3 or H3K27me3, highlighting that TADpole is able to distinguish functional TAD units. Additionally, we demonstrate that TADpole's hierarchical annotation, together with the new DiffT score, allows for detecting significant topological differences on Capture Hi-C maps between wild-type and genetically engineered mouse.European Research Council under the Seventh Framework Program FP7/2007-2013 [609989, in part]; European Union's Horizon 2020 Research and Innovation Programme [676556]; Spanish Ministry of Science and Innovation [BFU2013-47736-P, BFU2017-85926-P to M.A.M-R., IJCI-2015-23352 to I.F., BES-2014-070327 to P.S-V.]; ‘Centro de Excelencia Severo Ochoa 2013–2017’, SEV-2012-0208; CERCA Programme/Generalitat de Catalunya (to C.R.G.). Funding for open access charge: European Research Council under the Seventh Framework Program FP7/2007-2013 [609989]

Automatic analysis and 3D-modelling of Hi-C data using TADbit reveals structural features of the fly chromatin colors

The sequence of a genome is insufficient to understand all genomic processes carried out in the cell nucleus. To achieve this, the knowledge of its three-dimensional architecture is necessary. Advances in genomic technologies and the development of new analytical methods, such as Chromosome Conformation Capture (3C) and its derivatives, provide unprecedented insights in the spatial organization of genomes. Here we present TADbit, a computational framework to analyze and model the chromatin fiber in three dimensions. Our package takes as input the sequencing reads of 3C-based experiments and performs the following main tasks: (i) pre-process the reads, (ii) map the reads to a reference genome, (iii) filter and normalize the interaction data, (iv) analyze the resulting interaction matrices, (v) build 3D models of selected genomic domains, and (vi) analyze the resulting models to characterize their structural properties. To illustrate the use of TADbit, we automatically modeled 50 genomic domains from the fly genome revealing differential structural features of the previously defined chromatin colors, establishing a link between the conformation of the genome and the local chromatin composition. TADbit provides three-dimensional models built from 3C-based experiments, which are ready for visualization and for characterizing their relation to gene expression and epigenetic states. TADbit is an open-source Python library available for download from https://github.com/3DGenomes/tadbit.The research leading to these results has received funding from the European Research Council under the European Union's Seventh Framework Programme (FP7/2007-2013) / ERC grant agreement 609989, the Spanish Ministry of Economy and Competitiveness (BFU2013-47736-P) and the Human Frontiers Science Program (RGP0044). We acknowledge support of the CERCA Programme / Generalitat de Catalunya and the Spanish Ministry of Economy and Competitiveness, 'Centro de Excelencia Severo Ochoa 2013-2017', SEV-2012-0208 to the CRG