13 research outputs found

    Mining the human phenome using allelic scores that index biological intermediates

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    J. Kaprio ja M-L. Lokki työryhmien jäseniä.It is common practice in genome-wide association studies (GWAS) to focus on the relationship between disease risk and genetic variants one marker at a time. When relevant genes are identified it is often possible to implicate biological intermediates and pathways likely to be involved in disease aetiology. However, single genetic variants typically explain small amounts of disease risk. Our idea is to construct allelic scores that explain greater proportions of the variance in biological intermediates, and subsequently use these scores to data mine GWAS. To investigate the approach's properties, we indexed three biological intermediates where the results of large GWAS meta-analyses were available: body mass index, C-reactive protein and low density lipoprotein levels. We generated allelic scores in the Avon Longitudinal Study of Parents and Children, and in publicly available data from the first Wellcome Trust Case Control Consortium. We compared the explanatory ability of allelic scores in terms of their capacity to proxy for the intermediate of interest, and the extent to which they associated with disease. We found that allelic scores derived from known variants and allelic scores derived from hundreds of thousands of genetic markers explained significant portions of the variance in biological intermediates of interest, and many of these scores showed expected correlations with disease. Genome-wide allelic scores however tended to lack specificity suggesting that they should be used with caution and perhaps only to proxy biological intermediates for which there are no known individual variants. Power calculations confirm the feasibility of extending our strategy to the analysis of tens of thousands of molecular phenotypes in large genome-wide meta-analyses. We conclude that our method represents a simple way in which potentially tens of thousands of molecular phenotypes could be screened for causal relationships with disease without having to expensively measure these variables in individual disease collections.Peer reviewe

    Unconventional RNA-binding proteins step into the virus-host battlefront

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    The crucial participation of cellular RNA‐binding proteins (RBPs) in virtually all steps of virus infection has been known for decades. However, most of the studies characterizing this phenomenon have focused on well‐established RBPs harboring classical RNA‐binding domains (RBDs). Recent proteome‐wide approaches have greatly expanded the census of RBPs, discovering hundreds of proteins that interact with RNA through unconventional RBDs. These domains include protein–protein interaction platforms, enzymatic cores, and intrinsically disordered regions. Here, we compared the experimentally determined census of RBPs to gene ontology terms and literature, finding that 472 proteins have previous links with viruses. We discuss what these proteins are and what their roles in infection might be. We also review some of the pioneering examples of unorthodox RBPs whose RNA‐binding activity has been shown to be critical for virus infection. Finally, we highlight the potential of these proteins for host‐based therapies against viruses

    Identification of RNA-binding domains of RNA-binding proteins in cultured cells on a system-wide scale with RBDmap

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    RBDmap identifies, in a proteome-wide manner, the regions of RNA-binding proteins (RBPs) engaged in native interactions with RNA. In brief, cells are irradiated with UV light to induce protein-RNA crosslinks. Resulting covalently linked protein-RNA complexes are purified with oligo(dT) magnetic beads, following stringent denaturing washes. After elution, RBPs are subjected to partial proteolysis, where the protein regions still bound to the RNA and those released to the supernatant are separated by a second oligo (dT) selection. After sample preparation and mass spectrometric analysis, peptide intensity ratios between the RNA-bound and released fractions are used to determine the RNA-binding regions. As a Protocol Extension article, this article describes an adaptation of an existing Protocol, and offers additional applications. The earlier protocol (for the Interactome Capture method) describes how to identify the active RBPs of cultured cells, whilst this Protocol Extension enables the identification of the RNA-binding domains of RBPs in cultured cells. The experimental workflow takes one week, plus two additional weeks for proteomics and data analysis. Notably, RBDmap presents numerous advantages over classical methods to determine RNA-binding domains: it produces proteome-wide, high resolution maps of the protein regions contacting the RNA in a physiological context and can be adapted to different biological systems and conditions. Because RBDmap relies on the isolation of polyadenylated RNA via oligo (dT), it will not provide RNA-binding information on proteins interacting exclusively with non-polyadenylated transcripts. Applied to HeLa cells, RBDmap uncovered 1,174 RNA-binding sites in 529 proteins, many of which were previously unknown.

    Absolute quantitation of individual SARS-CoV-2 RNA molecules provides a new paradigm for infection dynamics and variant differences

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    Despite an unprecedented global research effort on SARS-CoV-2, early replication events remain poorly understood. Given the clinical importance of emergent viral variants with increased transmission, there is an urgent need to understand the early stages of viral replication and transcription. We used single-molecule fluorescence in situ hybridisation (smFISH) to quantify positive sense RNA genomes with 95% detection efficiency, while simultaneously visualising negative sense genomes, subgenomic RNAs, and viral proteins. Our absolute quantification of viral RNAs and replication factories revealed that SARS-CoV-2 genomic RNA is long-lived after entry, suggesting that it avoids degradation by cellular nucleases. Moreover, we observed that SARS-CoV-2 replication is highly variable between cells, with only a small cell population displaying high burden of viral RNA. Unexpectedly, the B.1.1.7 variant, first identified in the UK, exhibits significantly slower replication kinetics than the Victoria strain, suggesting a novel mechanism contributing to its higher transmissibility with important clinical implications
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