The Role of ATP and Cleavage Factor Phosphorylation in pre-mRNA 3\u27 Cleavage Reaction

The 3\u27 end cleavage/polyadenylation (3\u27 processing) is important in generating a functional messenger RNA (mRNA) transcript. It is long-known that ATP can significantly stimulate the in vitro cleavage of adenovirus type 2 L3 (Ad2L3) RNA substrate. Here, we used ATP analogs in structure-activity assays to show that the structural features of ATP and its analogs determine in vitro 3\u27 cleavage efficiency. More specifically, we demonstrate that the structure of the nitrogenous base, the nucleotide sugar and the triphosphate group contribute to the efficiency of the nucleotide-stimulated in vitro 3\u27 cleavage reaction. It was found that AppNp, an analog of ATP whose gamma phosphate cannot be transferred enzymatically or chemically to water (ATPase activity) and cannot serve as a phosphate donor for a protein kinase, is capable of stimulating cleavage. This finding led us to the conclusion that ATP likely functions as an allosteric activator of some protein involved in 3\u27 cleavage activity. The structure-activity relationship studies we carried out also showed that AppCp, another ATP analog whose gamma phosphate cannot be transferred, was an in vitro 3\u27 cleavage reaction inhibitor with an IC50 of approximately 300 μM. Taken together, our experiments provide indirect evidence that ATP (and certain analogs) stimulate 3\u27 cleavage by simply binding to one of the 3\u27 cleavage factors, and that their structures determine the efficiency of the 3\u27 cleavage reaction. Using the in vitro cleavage reaction, site-directed mutagenesis and co-immunoprecipitation (co-IP) we furthermore show that cleavage factors CFIm25, PAP and Clp1 are not likely to be the agencies of ATP action. From previous research in our lab we know that in vitro dephosphorylation of 3\u27 processing proteins, specifically cleavage factors I and II (denoted as CFm when unseparated), results in the suppression of the 3\u27 cleavage. We carried out experiments aiming to identify the phosphorylated cleavage factor(s) that can restore 3\u27 cleavage activity to dephosphorylated HeLa cell nuclear extract (NxT). It was determined that immunopurified Flag epitope-tagged-CFIm25, -Clp1, -CFIm68, -CFIm59, and tag-independent immunopurified Pcf11, when used in place of CFm, were not capable of restoring 3\u27 cleavage activity to dephosphorylated NxT, and therefore likely do not contain an activating amount of the unidentified phosphorylated cleavage factor

Human germline heterozygous gain-of-function STAT6 variants cause severe allergic disease

STAT6 (signal transducer and activator of transcription 6) is a transcription factor that plays a central role in the pathophysiology of allergic inflammation. We have identified 16 patients from 10 families spanning three continents with a profound phenotype of early-life onset allergic immune dysregulation, widespread treatment-resistant atopic dermatitis, hypereosinophilia with esosinophilic gastrointestinal disease, asthma, elevated serum IgE, IgE-mediated food allergies, and anaphylaxis. The cases were either sporadic (seven kindreds) or followed an autosomal dominant inheritance pattern (three kindreds). All patients carried monoallelic rare variants in STAT6 and functional studies established their gain-of-function (GOF) phenotype with sustained STAT6 phosphorylation, increased STAT6 target gene expression, and TH2 skewing. Precision treatment with the anti-IL-4Rα antibody, dupilumab, was highly effective improving both clinical manifestations and immunological biomarkers. This study identifies heterozygous GOF variants in STAT6 as a novel autosomal dominant allergic disorder. We anticipate that our discovery of multiple kindreds with germline STAT6 GOF variants will facilitate the recognition of more affected individuals and the full definition of this new primary atopic disorder

Microbiome connections with host metabolism and habitual diet from 1,098 deeply phenotyped individuals

The gut microbiome is shaped by diet and influences host metabolism; however, these links are complex and can be unique to each individual. We performed deep metagenomic sequencing of 1,203 gut microbiomes from 1,098 individuals enrolled in the Personalised Responses to Dietary Composition Trial (PREDICT 1) study, whose detailed long-term diet information, as well as hundreds of fasting and same-meal postprandial cardiometabolic blood marker measurements were available. We found many significant associations between microbes and specific nutrients, foods, food groups and general dietary indices, which were driven especially by the presence and diversity of healthy and plant-based foods. Microbial biomarkers of obesity were reproducible across external publicly available cohorts and in agreement with circulating blood metabolites that are indicators of cardiovascular disease risk. While some microbes, such as Prevotella copri and Blastocystis spp., were indicators of favorable postprandial glucose metabolism, overall microbiome composition was predictive for a large panel of cardiometabolic blood markers including fasting and postprandial glycemic, lipemic and inflammatory indices. The panel of intestinal species associated with healthy dietary habits overlapped with those associated with favorable cardiometabolic and postprandial markers, indicating that our large-scale resource can potentially stratify the gut microbiome into generalizable health levels in individuals without clinically manifest disease