Dataset Information

System-wide profiling of protein-RNA interactions in sindbis infected cells reveals host regulators of infection

ABSTRACT: RNA viruses rely on cellular RNA-binding proteins (RBPs) to infect the host cell. However, the repertoire of the cellular RBPs exploited by viruses is largely unknown. Using the ‘RNA interactome capture’ method, we profiled in a proteome-wide scale the RBP-RNA interactions occurring during sindbis virus (SINV) infection. We discover that SINV affects the activity of hundreds of RBPs, essentially rewiring the host RNA-bound proteome. Such alterations do not relate to changes in protein abundance but are rather due to subcellular redistribution of RBPs and pervasive transcriptome remodelling. Strikingly, RBPs stimulated by SINV accumulate in the cytoplasmic compartments where the virus replicates and interact with viral RNA. Perturbation of these RBPs can restrict or promote infection; for example, GEMIN5 binds to key regulatory regions of SINV RNAs and inhibits viral protein expression. Importantly, we show that drug based RBP intervention may have potential as therapeutic strategy against viruses.

OTHER RELATED OMICS DATASETS IN: GSE125182

INSTRUMENT(S): LTQ Orbitrap Elite, Q Exactive

ORGANISM(S): Sindbis Virus Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Caroline Lenz

LAB HEAD: Alfredo Castello Palomares

PROVIDER: PXD009789 | Pride | 2019-02-28

REPOSITORIES: Pride

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Publications

System-wide Profiling of RNA-Binding Proteins Uncovers Key Regulators of Virus Infection.

Garcia-Moreno Manuel M Noerenberg Marko M Ni Shuai S Järvelin Aino I AI González-Almela Esther E Lenz Caroline E CE Bach-Pages Marcel M Cox Victoria V Avolio Rosario R Davis Thomas T Hester Svenja S Sohier Thibault J M TJM Li Bingnan B Heikel Gregory G Michlewski Gracjan G Sanz Miguel A MA Carrasco Luis L Ricci Emiliano P EP Pelechano Vicent V Davis Ilan I Fischer Bernd B Mohammed Shabaz S Castello Alfredo A

Molecular cell 20190221 1

The compendium of RNA-binding proteins (RBPs) has been greatly expanded by the development of RNA-interactome capture (RIC). However, it remained unknown if the complement of RBPs changes in response to environmental perturbations and whether these rearrangements are important. To answer these questions, we developed "comparative RIC" and applied it to cells challenged with an RNA virus called sindbis (SINV). Over 200 RBPs display differential interaction with RNA upon SINV infection. These alte ...[more]

PMID: 30799147

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Project description:RNA-binding proteins (RBPs) are intimately involved in all aspects of RNA processing and regulation and are linked to neurodegenerative diseases and cancer. Therefore, understanding the relationship between RBPs and their RNA targets is critical for a broader understanding of post-transcriptional regulation in normal and disease processes. The majority of approaches to study RNA-protein interactions focus on single RBPs, however there are many hundreds RBPs encoded in the human genome, and each cell type expresses a different catalog of these regulatory molecules, greatly limiting the ability of these single RBP approaches to capture the global landscape of RNA-protein interactions. We and others have developed approaches to globally catalog regions of mRNAs that are bound by proteins in an unbiased manner. Here we describe a detailed protocol for performing our RNase-mediated protein footprint sequencing approach, termed protein interaction profile sequencing (PIP-seq). In this protocol, RNA-protein interactions are stabilized by cross-linking, and un-bound regions are digested with RNases, leaving only the protein-bound regions intact. To control for RNase insensitive regions, proteins are first denatured and then RNP complexes are subject to RNase treatment. After high-throughput sequencing of remaining fragments, peak calling is performed to identify protein protected sites (PPSs). We describe the application of this protocol to a human embryonic kidney cell line and perform basic quality control, reproducibility and benchmarking analyses. Finally, we describe the landscape of protein-interactions in HEK293T cells, underscoring the value of this approach. Future applications of this method to study the dynamics of RNA-protein interactions in developmental processes will help to uncover the role of RBPs in post-transcriptional regulation. Protein interaction profile sequencing (PIP-seq) in HEK293T cells. Three replicates of formaldehyde cross-linked PIP-seq are described

Dataset Information

System-wide profiling of protein-RNA interactions in sindbis infected cells reveals host regulators of infection

Publications

System-wide Profiling of RNA-Binding Proteins Uncovers Key Regulators of Virus Infection.

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