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Inorganic pyrophosphatase (iPPase) is an enzyme that cleaves pyrophosphate into two phosphate molecules. This enzyme is an essential component of in vitro transcription (IVT) reactions for RNA preparation as it prevents pyrophosphate from precipitating with magnesium, ultimately increasing the rate of the IVT reaction. Large-scale RNA production is often required for biochemical and biophysical characterization studies of RNA, therefore requiring large amounts of IVT reagents. Commercially purchased iPPase is often the most expensive component of any IVT reaction. In this paper, we demonstrate that iPPase can be produced in large quantities and high quality using a reasonably generic laboratory facility and that laboratory-purified iPPase is as effective as commercially available iPPase. Furthermore, using size exclusion chromatography coupled with multi-angle light scattering and dynamic light scattering, analytical ultracentrifugation, and small-angle X-ray scattering, we demonstrate that yeast iPPase can form tetramers and hexamers in solution as well as the enzymatically active dimer. Our work provides a robust protocol for laboratories involved with RNA in vitro transcription to efficiently produce active iPPase, significantly reducing the financial strain of large-scale RNA production. Key words: inorganic pyrophosphatase, RNA in vitro transcription, iPPase, analytical ultracentrifuge, small-angle X-ray scattering La pyrophosphatase inorganique (iPPase) est une enzyme qui clive le pyrophosphate en deux molécules de phosphate. Cette enzyme est un composant essentiel des réactions de transcription in vitro (TIV) pour la préparation d'ARN, car elle empêche le pyrophosphate de précipiter avec le magnésium, ce qui augmente finalement la vitesse de la réaction de TIV. La production d'ARN à grande échelle est souvent requise pour les études de caractérisation biochimique et biophysique de i'ARN, ce qui nécessite de grandes quantités de réactifs pour la TIV. L'iPPase achetée dans le commerce est souvent le composant le plus cher de toute réaction de TIV. Dans cet article, les auteurs démontrent que l'iPPase peut être produite en grandes quantités avec une haute qualité en utilisant une installation de laboratoire raisonnablement générique, et que l'iPPase purifiée en laboratoire est aussi efficace que l'iPPase disponible dans le commerce. De plus, en utilisant la chromatographie d'exclusion stérique couplée à la diffusion multiangle de la lumière et à la diffusion dynamique de la lumière, l'ultracentrifugation analytique et la diffusion de rayons X aux petits angles, ils démontrent que l'iPPase de levure peut former des tétramères et des hexamères en solution, ainsi que le dimère enzymatiquement actif. Ce travail offre un protocole robuste pour les laboratoires engagés dans la transcription in vitro d'ARN afin de produire efficacement l'iPPase active, réduisant ainsi de manière significative les contraintes financières de la production d'ARN à grande échelle. [Traduit par la Rédaction] Mots-clés : pyrophosphatase inorganique, transcription in vitro de l'ARN, iPPase, ultracentrifugeuse analytique, diffusion des rayons X aux petits angles, Introduction Inorganic pyrophosphatase (iPPase; EC 3.6.1.1) is primarily characterized by its function to catalyze the hydrolysis of pyrophosphate into two monophosphates (Harold 1966), as presented in eq. 1. Three different [...]

Co‐existing chronic hepatitis B virus (CHB) infection and metabolic dysfunction associated steatotic liver disease (MASLD) can exert complex effects on hepatic metabolism, requiring mechanistic study. CHB participants were assessed for MASLD and the impact of hepatic steatosis/metabolic syndrome (MetS) on novel viral and immunological markers. In this prospective, cohort study, untreated CHB subjects were assessed for liver disease by non‐invasive tests (i.e. FibroScan, controlled attenuation parameter, CAP). Subjects were tested for cytokines and IFN‐γ ELISPOT assay to HBV Surface (S) and Core (C) proteins. Standard HBV serological, exploratory biomarkers and deep sequencing of HBV S and C genes were performed. In 53 subjects (median age 45 years [SD = 10.6], 35% F, 56% Asian, 20% Black, 3% White), 94% (50) HBeAg negative, 63% genotype B/C, mean HBV DNA 3.2 log10 IU/mL (SD = 1.8), quantitative HBsAg 2.9 log10 IU/mL (SD = 1.2) and HBV pgRNA 2.1 log10 copies/mL (SD = 1.3). In enrolled subjects, the mean ALT was 41.9 U/L (SD = 24.0), FibroScan was 5.7 kPa (SD = 1.9) and CAP was 306.4 dB/m (SD = 49.0). The mean BMI was 28.2 kg/m2 (SD = 4.2), 20% (11/53) had diabetes, 35% (19/53) dyslipidaemia and 24% (13/53) hypertension. Subjects with MetS and steatosis showed lower HBV markers (p <.01), higher HBV S diversity (p =.02) and greater frequency of HBV variants associated with host‐anti‐viral immune escape. Pro‐inflammatory cytokine levels and HBV‐specific cellular responses were higher in participants with hepatic steatosis. In CHB, MASLD/hepatic steatosis was associated with HBV variants and systemic immune responses potentially impacting liver disease progression despite low‐level viraemia. [ABSTRACT FROM AUTHOR]

RNA is involved in all domains of life, playing critical roles in a host of gene expression processes, host-defense mechanisms, cell proliferation, and diseases. A critical component in many of these events is the ability for RNA to interact with proteins. Over the past few decades, our understanding of such RNA--protein interactions and their importance has driven the search and development of new techniques for the identification of RNA-binding proteins. In determining which proteins bind to the RNA of interest, it is often useful to use the approach where the RNA molecule is the 'bait' and allow it to capture proteins from a lysate or other relevant solution. Here, we review a collection of methods for modifying RNA to capture RNA-binding proteins. These include small-molecule modification, the addition of aptamers, DNA-anchoring, and nucleotide substitution. With each, we provide examples of their application, as well as highlight their advantages and potential challenges. Key words: RNA-binding proteins, biotin labelling, digoxigenin labelling, aptamer labelling, affinity capture, nucleotide-substitution method. L'ARN est implique dans tous les domaines de la vie, jouant un role cle dans nombre de processus d'expression genique, de mecanismes de defense de l'hote, dans la proliferation cellulaire et les maladies. Une composante importante de plusieurs de ces manifestations consiste en la capacite de l'ARN a interagir avec les proteines. Au cours des quelques dernieres decennies, notre comprehension de telles interactions ARN--proteines et de leur importance a stimule la recherche et le developpement de nouvelles techniques visant l'identification de proteines liant l'ARN. Afin de determiner quelles proteines se lient a un ARN d'interet, il est souvent utile d'utiliser une approche ou la molecule d'ARN sert d'appat, lui permettant de capturer les proteines d'un lysat ou d'une autre solution pertinente. Les auteurs font ici la synthese d'une serie de methodes pour modifier l'ARN afin de capturer des proteines liant l'ARN. Celles-ci comprennent la modification de petites molecules, l'ajout d'aptameres, l'ancrage d'ADN et la substitution de nucleotides. Pour chacune, ils fournissent des exemples de leur application et soulignent leurs avantages et defis potentiels. [Traduit par la Redaction] Mots-cles : proteines liant l'ARN, etiquetage a la biotine, etiquetage a la digoxigenine, etiquetage d'aptameres, capture d'affinite, methode de substitution de nucleotides., Introduction Over the past several decades, great strides have been made in characterizing the roles of many types of RNA-binding proteins. This is due to the development of newer technologies, [...]

To achieve a virological cure for hepatitis B virus (HBV), innovative strategies are required to target the covalently closed circular DNA (cccDNA) genome. Guanine‐quadruplexes (G4s) are a secondary structure that can be adopted by DNA and play a significant role in regulating viral replication, transcription, and translation. Antibody‐based probes and small molecules have been developed to study the role of G4s in the context of the human genome, but none have been specifically made to target G4s in viral infection. Herein, we describe the development of a humanized single‐domain antibody (S10) that can target a G4 located in the PreCore (PreC) promoter of the HBV cccDNA genome. MicroScale Thermophoresis demonstrated that S10 has a strong nanomolar affinity to the PreC G4 in its quadruplex form and a structural electron density envelope of the complex was determined using Small‐Angle X‐ray Scattering. Lentiviral transduction of S10 into HepG2‐NTCP cells shows nuclear localization, and chromatin immunoprecipitation coupled with next‐generation sequencing demonstrated that S10 can bind to the HBV PreC G4 present on the cccDNA. This research validates the existence of a G4 in HBV cccDNA and demonstrates that this DNA secondary structure can be targeted with high structural and sequence specificity using S10. [ABSTRACT FROM AUTHOR]

The RiboWest Conference brings together RNA researchers in Canada with the 2-fold goals of fostering internationally competitive RNA research and of training the next generation of scientists. The 14th Annual RiboWest conference (RiboWest 2018) was held at the University of Lethbridge (Lethbridge, Alberta) from June 10th to 13th, 2018. This meeting was focused on all major aspects of RNA research, ranging from understanding the cellular role of RNA, studying RNA interactions and structures, and employing them as a therapeutic tool. The invited keynote speakers (5) provided insights into the wide-range of RNA-based research. One of the unique features of this conference was that the majority of the oral presentations were given by the trainees (undergraduate/graduate students and postdoctoral researchers). Hosted by the Alberta RNA Research and Training Institute (ARRTI) at the University of Lethbridge as the leading center of RNA research in Western Canada, the RiboWest 2018 was well attended by researchers from across the country (>110 attendees in total). This conference proceedings editorial presents the overview of the conference, and briefly introduces articles published in this special issue of Biochemistry and Cell Biology. Key words: RiboWest, RNA, Western Canadian RNA Conference, University of Lethbridge. La RiboWest Conference reunit annuellement des chercheurs oeuvrant dans le domaine de l'ARN au Canada, avec le double objectifdepromouvoir une recherche sur l'ARNde calibre mondialetde former laprochaine generationde scientifiques. La [14.sup.e] RiboWest Conference (RiboWest 2018) s'est tenue a l'Universite de Lethbridge (Lethbridge, Alberta) du 10 au 13 juin 2018. Ce colloque etait consacre a tous les principaux aspects de la recherche sur l'ARN, allant de la comprehension du role des ARN dans la cellule, l'etude des interactions et des structures des ARN et leur utilisation comme outils therapeutiques. Les cinq conferenciers d'honneur ont offert un apercu de la vaste etendue de la recherche sur l'ARN. Une des particularites de ce colloque venait du fait que la majorite des presentations orales ont ete donnees par des stagiaires (etudiants de premier cycle, etudiants de cycles superieurs et chercheurs postdoctoraux). Accueilli par le Alberta RNA Research and Training Institute (ARRTI) de l'Universite de Lethbridge en sa qualite de centre de pointe de la recherche sur l'ARN dans l'Ouest canadien, la RiboWest 2018 a rassemble des chercheurs de tout le pays (>110 participants au total). Cet editorial des actes du colloque presente une vue d'ensemble du colloque et introduit brievement les articles publies dans ce numero special de la revue Biochimie et biologie cellulaire. [Traduit par la Redaction] Mots-cles : RiboWest, ARN, Western Canada RNA Conference, Universite de Lethbridge., Introduction The first RiboWest conference was held in 2005 at the University of Northern British Columbia, British Columbia, Canada, spearheading the formation of a collaborative community of RNA researchers in [...]