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RNA-binding proteins (RBPs) are critical effectors of gene expression, and as such their malfunction underlies the origin of many diseases. RBPs can recognize hundreds of transcripts and form extensive regulatory networks that help to maintain cell homeostasis. System-wide unbiased identification of RBPs has increased the number of recognized RBPs into the four-digit range and revealed new paradigms: from the prevalence of structurally disordered RNA-binding regions with roles in the formation of membraneless organelles to unsuspected and potentially pervasive connections between intermediary metabolism and RNA regulation. Together with an increasingly detailed understanding of molecular mechanisms of RBP function, these insights are facilitating the development of new therapies to treat malignancies. Here, we provide an overview of RBPs involved in human genetic disorders, both Mendelian and somatic, and discuss emerging aspects in the field with emphasis on molecular mechanisms of disease and therapeutic interventions. ; The authors acknowledge funding from the Spanish Ministry of Science and Innovation (MICINN; PGC2018-099697-B-I00 to F.G. and BFU2017-89308-P to J.V.), 'la Caixa' Foundation (ID 100010434 under the agreement LCF/PR/HR17/52150016 to F.G.), the Catalan Government (2017SGR534 to F.G. and J.V.) and the ERC (AdvG 670146 to J.V.) as well as the EMBL Partnership, the Severo Ochoa and CERCA Programs

Lonneke van der Linden et al. ; The genus Enterovirus of the family Picornaviridae contains many important human pathogens (e.g., poliovirus, coxsackievirus, rhinovirus, and enterovirus 71) for which no antiviral drugs are available. The viral RNA-dependent RNA polymerase is an attractive target for antiviral therapy. Nucleoside-based inhibitors have broad-spectrum activity but often exhibit off-target effects. Most non-nucleoside inhibitors (NNIs) target surface cavities, which are structurally more flexible than the nucleotide-binding pocket, and hence have a more narrow spectrum of activity and are more prone to resistance development. Here, we report a novel NNI, GPC-N114 (2,2'-[(4-chloro-1,2-phenylene)bis(oxy)]bis(5-nitro-benzonitrile)) with broad-spectrum activity against enteroviruses and cardioviruses (another genus in the picornavirus family). Surprisingly, coxsackievirus B3 (CVB3) and poliovirus displayed a high genetic barrier to resistance against GPC-N114. By contrast, EMCV, a cardiovirus, rapidly acquired resistance due to mutations in 3Dpol. In vitro polymerase activity assays showed that GPC-N114 i) inhibited the elongation activity of recombinant CVB3 and EMCV 3Dpol, (ii) had reduced activity against EMCV 3Dpol with the resistance mutations, and (iii) was most efficient in inhibiting 3Dpol when added before the RNA template-primer duplex. Elucidation of a crystal structure of the inhibitor bound to CVB3 3Dpol confirmed the RNA-binding channel as the target for GPC-N114. Docking studies of the compound into the crystal structures of the compound-resistant EMCV 3Dpol mutants suggested that the resistant phenotype is due to subtle changes that interfere with the binding of GPC-N114 but not of the RNA template-primer. In conclusion, this study presents the first NNI that targets the RNA template channel of the picornavirus polymerase and identifies a new pocket that can be used for the design of broad-spectrum inhibitors. Moreover, this study provides important new insight into the plasticity of picornavirus polymerases at the template binding site. © 2015 van der Linden et al. ; This work was supported by research grants from the SILVER Large Scale Collaborative Project (grant agreement number 260644) project of the European Union 7th Framework, the European Virus Archive (EVA) project (European FP7 Capacities Project number 153 228292), the "Convenant K.U. Leuven-Radboud University Nijmegen" framework, GOA/10/014 (for work performed at KULeuven), NWO-VICI grant no. 91812628 (FJMvK), FWO Krediet aan Navorsers no. 1.5.206.11 (AMDP), the "Agency for Innovation by Science and Technology in Flanders (IWT)" (CL) ; Peer Reviewed

MOTIVATION: The avalanche of data arriving since the development of NGS technologies have prompted the need for developing fast, accurate and easily automated bioinformatic tools capable of dealing with massive datasets. Among the most productive applications of NGS technologies is the sequencing of cellular RNA, known as RNA-Seq. Although RNA-Seq provides similar or superior dynamic range than microarrays at similar or lower cost, the lack of standard and user-friendly pipelines is a bottleneck preventing RNA-Seq from becoming the standard for transcriptome analysis. RESULTS: In this work we present a pipeline for processing and analyzing RNA-Seq data, that we have named Grape (Grape RNA-Seq Analysis Pipeline Environment). Grape supports raw sequencing reads produced by a variety of technologies, either in FASTA or FASTQ format, or as prealigned reads in SAM/BAM format. A minimal Grape configuration consists of the file location of the raw sequencing reads, the genome of the species and the corresponding gene and transcript annotation. Grape first runs a set of quality control steps, and then aligns the reads to the genome, a step that is omitted for prealigned read formats. Grape next estimates gene and transcript expression levels, calculates exon inclusion levels and identifies novel transcripts. Grape can be run on a single computer or in parallel on a computer cluster. It is distributed with specific mapping and quantification tools, but given its modular design, any tool supporting popular data interchange formats can be integrated. AVAILABILITY: Grape can be obtained from the Bioinformatics and Genomics website at: http://big.crg.cat/services/grape. ; The research leading to these results has received funding from the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement 282510. This work has been carried under grants BIO2011-26205 from Ministerio de Economía y Competitividad (Spain) and INB GNV-1 and RETICS RD07/0067/0012 from PN de I+D+i, ISCIII—Subdirección General de Evaluación y Fomento de la Investigación—(Spain) and cofunded by FEDER

In den letzten Jahren sorgte das humanpathogene Ebola-Virus (EBOV) vermehrt für Epidemien vor allem in Zentral- und Westafrika mit Letalitätsraten von bis zu 90 %. Wirksame antivirale Kausaltherapien sind bis dato nicht verfügbar. Aktuell ist die Demokratische Republik Kongo von einer schweren EBOV-Epidemie betroffen. Das EBOV-Genom ist eine nicht-segmentierte Negativstrang (NNS)-RNA. An den terminalen 3'- und 5'-Genomenden befinden sich regulatorische Elemente der EBOVReplikation und -Transkription (3'-Leader, 5'-Trailer). Die EBOV-Replikation wird durch einen Proteinkomplex aus drei viralen Proteinen gesteuert, der RNA-abhängigen RNA-Polymerase L, ihrem Kofaktor, dem viralen Protein 35 (VP35), und dem Nukleoprotein (NP). Die virale Transkription benötigt darüber hinaus das virale Protein 30 (VP30) als Transkriptionsaktivator. Vermutlich nutzt die Virus-Polymerase eine einzige Eintrittsstelle am 3'-Genomende, von der aus sowohl Replikation als auch Transkription initiiert werden. Im Falle anderer NNS-Viren führt dies zur Synthese eines kurzen Abbruchtranskripts antisense zum 3'-Leader vor der Initiation der mRNA-Synthese am ersten Genstartsignal. Der genomische Replikationspromoter ist zweigeteilt. Promoterelemente 1 und 2 (PE1, PE2) sind durch die Transkriptionsstartsequenz (TSS) und weitere Platzhalternukleotide (Spacer) getrennt. Entsprechend der "Rule of 6" darf der Spacer lediglich um ein Vielfaches der Zahl 6 erweitert oder verkürzt werden, um eine effiziente Replikation zu gewährleisten. PE2 weist darüber hinaus 8 konsekutive 3'-UN5-Hexamere auf, von denen 3 obligat für eine residuale Replikationsaktivität sind. Auch der TSS-Spacer-Bereich enthält UN5-Hexamere. Diese sind jedoch an Nt -75 durch ein Guanosin anstelle eines Uridins vom PE2-UN5-Raster getrennt. Der Spacer kann sowohl auf genomischer als auch auf der komplementären mRNA-Ebene putative Haarnadelstrukturen unter Einbeziehung der TSS ausbilden (NP-Hairpin). Diesem NP-Hairpin wird eine Rolle in der VP30-abhängigen Transkriptionsregulation zugeschrieben. Ziel der vorliegenden Studie war es, ein besseres Verständnis der Polymerisationsinitiation am 3'-Leader-Promotor zu erlangen. Im Fokus standen neben der Untersuchung der Replikations- und Transkriptionspromotorarchitektur die Analyse regulatorischer RNA-Sequenzen, die Rolle der TSS-Hairpin-Struktur sowie die Regulation der Transkription durch VP30. Mithilfe einer gezielten Verwendung Replikations-kompetenter und Replikations-defizienter Minigenomvarianten in Kombination mit Reportergenassays und qRT-PCR konnten wir demonstrieren, dass PE1 ebenfalls den Transkriptionspromotor kodiert. Darüber hinaus zeigen unsere Daten, dass die "Rule of 6" in der Spacer-Region nicht nur für die Replikations- sondern auch für die Transkriptionsinitiation relevant ist. Eine Verlängerung des Spacer-Bereichs ist, abhängig vom Sequenz- und Strukturkontext, um mindestens 66 Nt ohne vollständigen Funktionsverlust möglich. Die Funktion der UN5-Hexamere in Spacer und PE2 ist nach wie vor spekulativ. Möglicherweise positionieren die Hexamere das Nukleoprotein während der RNA-Synthese in spezifischer Anordnung auf dem RNA-Templat, um eine produktive Promotorerkennung durch die virale Polymerase zu ermöglichen, oder sie koordinieren die NP-Dissoziation/Reassoziation am RNA-Templatstrang, während die RNA durch das aktive Zentrum der Polymerase gefädelt wird. Unsere Daten deuten darauf hin, dass ein kontinuierliches UN5-Hexamerraster zwischen PE1 und PE2 aufgrund einer G-75 zu U-Punktmutation die allgemeine Effizienz von Transkription und Replikation steigert, jedoch auf Kosten der selektiven Feinregulation beider Prozesse durch VP30 (Transkriptionsaktivierung und Replikationsrepression). Im Fall einer Spacer-Deletion von 12 Nt, die gleichzeitig die Ausbildung des NP-Hairpins verhindert, ist die Replikations- und Transkriptionsaktivität nach wie vor > 50 % und die Regulation durch VP30 bleibt grundsätzlich erhalten. Dies verdeutlicht, dass Sekundärstrukturen am Transkriptionsstart weder essenziell für die VP30-abhängige Transkription noch für die Replikation sind. Allerdings konnten wir zeigen, dass Hairpindestabilisierungen tendenziell die VP30-Abhängigkeit der Transkription relaxieren, während Stabilisierungen diese steigern. Eine schrittweise Stabilisierung der TSS-Hairpinstruktur durch strategische Insertion von G:C-Basenpaaren und die damit verbundene Ausweitung lokaler Doppelstrangbereiche führte, abhängig vom Ausmaß der Stabilisierung auf Antigenom/mRNA-Ebene, zu starken Aktivitätsverlusten bis hin zur Elimination der Transkriptions- als auch Replikationsaktivität. Damit konnten wir zum ersten Mal indirekt die Ausbildung von RNA-Strukturen während der viralen RNA-Synthese nachweisen. Allgemein führten jedwede Änderungen der Sequenz, Struktur, Stabilität oder Länge des nativen NP-Hairpins zu einer partiell verringerten VP30- Abhängigkeit der Transkription. Somit erscheint der NP-Hairpin optimiert für eine engmaschige Regulation durch VP30. Hochdurchsatz-RNA-Sequenzierung und Northern Blot-Analyse bestätigten darüber hinaus die Synthese abortiver leaderRNAs. Ihre Initiation erfolgte wie im Fall der Antigenomsynthese ausschließlich an Nt -2. Im Gegensatz dazu sorgte der Erhalt des ersten Genomnukleotids für eine Vervierfachung der Transkription. Sowohl die leaderRNA-Initiation an Nt -2 als auch die Termination in einem Längenbereich von ~ 60 - 80 Nt erfolgten unabhängig von RNA-Sequenz/-Struktur oder von VP30. In Anwesenheit von VP30 sind die leaderRNA-Spiegel allgemein reduziert. 24 h nach EBOV-Infektion bzw. 48 h nach Minigenom-Transfektion sind die leaderRNA-Spiegel 9-fach bzw. 68-fach geringer als die der ersten mRNA. Zusammenfassend deuten unsere Daten darauf hin, dass leaderRNAs Abbruchprodukte der Antigenomsynthese und möglicherweise keine obligaten Vorlauf-Produkte der viralen Transkription sind.

Human noroviruses in the family Caliciviridae are a major cause of epidemic gastroenteritis. They are responsible for at least 95% of viral outbreaks and over 50% of all outbreaks worldwide. Transmission of these highly infectious plus-stranded RNA viruses occurs primarily through contaminated food or water, but also through person-to-person contact and exposure to fomites. Norovirus infections are typically acute and self-limited. However, disease can be much more severe and prolonged in infants, elderly, and immunocompromised individuals. Norovirus outbreaks frequently occur in semi-closed communities such as nursing homes, military settings, schools, hospitals, cruise ships, and disaster relief situations. Noroviruses are classified as Category B biodefense agents because they are highly contagious, extremely stable in the environment, resistant to common disinfectants, and associated with debilitating illness. The number of reported norovirus outbreaks has risen sharply since 2002 suggesting the emergence of more infectious strains. There has also been increased recognition that noroviruses are important causes of childhood hospitalization. Moreover, noroviruses have recently been associated with multiple clinical outcomes other than gastroenteritis. It is unclear whether these new observations are due to improved norovirus diagnostics or to the emergence of more virulent norovirus strains. Regardless, it is clear that human noroviruses cause considerable morbidity worldwide, have significant economic impact, and are clinically important emerging pathogens. Despite the impact of human norovirus-induced disease and the potential for emergence of highly virulent strains, the pathogenic features of infection are not well understood due to the lack of a cell culture system and previous lack of animal models. This review summarizes the current understanding of norovirus pathogenesis from the histological to the molecular level, including contributions from new model systems.

One of the challenges being faced in the twenty-first century is the biological control of plant viral infections. Among the different strategies to combat virus infections, those based on pathogen-derived resistance (PDR) are probably the most powerful approaches to confer virus resistance in plants. The application of the PDR concept not only revealed the existence of a previously unknown sequence-specific RNA-degradation mechanism in plants, but has also helped to design antiviral strategies to engineer viral resistant plants in the last 25 years. In this article, we review the different platforms related to RNA silencing that have been developed during this time to obtain plants resistant to viruses and illustrate examples of current applications of RNA silencing to protect crop plants against viral diseases of agronomic relevance. This article is part of a Special Issue entitled: MicroRNAs in viral gene regulation. © 2011. ; The work in the author's laboratory is supported by grants BIO2010-18541 from Spanish MEC and KBBE-204429 from European Union. ; Peer Reviewed

Human noroviruses in the family Caliciviridae are a major cause of epidemic gastroenteritis. They are responsible for at least 95% of viral outbreaks and over 50% of all outbreaks worldwide. Transmission of these highly infectious plus-stranded RNA viruses occurs primarily through contaminated food or water, but also through person-to-person contact and exposure to fomites. Norovirus infections are typically acute and self-limited. However, disease can be much more severe and prolonged in infants, elderly, and immunocompromised individuals. Norovirus outbreaks frequently occur in semi-closed communities such as nursing homes, military settings, schools, hospitals, cruise ships, and disaster relief situations. Noroviruses are classified as Category B biodefense agents because they are highly contagious, extremely stable in the environment, resistant to common disinfectants, and associated with debilitating illness. The number of reported norovirus outbreaks has risen sharply since 2002 suggesting the emergence of more infectious strains. There has also been increased recognition that noroviruses are important causes of childhood hospitalization. Moreover, noroviruses have recently been associated with multiple clinical outcomes other than gastroenteritis. It is unclear whether these new observations are due to improved norovirus diagnostics or to the emergence of more virulent norovirus strains. Regardless, it is clear that human noroviruses cause considerable morbidity worldwide, have significant economic impact, and are clinically important emerging pathogens. Despite the impact of human norovirus-induced disease and the potential for emergence of highly virulent strains, the pathogenic features of infection are not well understood due to the lack of a cell culture system and previous lack of animal models. This review summarizes the current understanding of norovirus pathogenesis from the histological to the molecular level, including contributions from new model systems.