Dataset Information

IrCLIP platform for efficient characterization of protein-RNA interactions.

ABSTRACT: The complexity of transcriptome-wide protein-RNA interaction networks is incompletely understood. While emerging studies are greatly expanding the known universe of RNA-binding proteins, methods for the discovery and characterization of protein-RNA interactions remain resource intensive and technically challenging. Here we introduce a UV-C crosslinking and immunoprecipitation platform, irCLIP, which provides an ultraefficient, fast, and nonisotopic method for the detection of protein-RNA interactions using far less material than standard protocols.

SUBMITTER: Zarnegar BJ

PROVIDER: S-EPMC5477425 | biostudies-literature | 2016 Jun

REPOSITORIES: biostudies-literature

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irCLIP platform for efficient characterization of protein-RNA interactions.

Zarnegar Brian J BJ Flynn Ryan A RA Shen Ying Y Do Brian T BT Chang Howard Y HY Khavari Paul A PA

Nature methods 20160425 6

The complexity of transcriptome-wide protein-RNA interaction networks is incompletely understood. While emerging studies are greatly expanding the known universe of RNA-binding proteins, methods for the discovery and characterization of protein-RNA interactions remain resource intensive and technically challenging. Here we introduce a UV-C crosslinking and immunoprecipitation platform, irCLIP, which provides an ultraefficient, fast, and nonisotopic method for the detection of protein-RNA interac ...[more]

PMID: 27111506

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Project description:Arthropod-borne viruses, such as the members of the genus Alphavirus, are a significant concern to global public health. As obligate intracellular pathogens, RNA viruses must interact with the host cell machinery to establish and complete their life cycles. Despite considerable efforts to define the host-pathogen interactions essential for alphaviral replication, an unbiased and inclusive assessment of alphaviral RNA-protein interactions has not been undertaken. Moreover, the biological and molecular importance of these interactions, in the full context of their molecular function as RNA-binding proteins, has not been fully realized. The data presented here introduce a robust viral RNA-protein discovery method to elucidate the Sindbis virus (SINV) RNA-protein host interface. Cross-link-assisted mRNP purification (CLAMP) assessment revealed an extensive array of host-pathogen interactions centered on the viral RNAs (vRNAs). After prioritization of the host proteins associated with the vRNAs, we identified the site of protein-vRNA interaction by a UV cross-linking and immunoprecipitation sequencing (CLIP-seq) approach and assessed the consequences of the RNA-protein binding event of hnRNP K, hnRNP I, and hnRNP M in regard to viral infection. Here, we demonstrate that mutation of the prioritized hnRNP-vRNA interaction sites effectively disrupts hnRNP-vRNA interaction. Correlating with disrupted hnRNP-vRNA binding, SINV growth kinetics were reduced relative to wild-type parental viral infections in vertebrate and invertebrate tissue culture models of infection. The molecular mechanism leading to reduced viral growth kinetics was found to be dysregulated structural-gene expression. Collectively, this study further defines the scope and importance of the alphavirus host-pathogen vRNA-protein interactions.IMPORTANCE Members of the genus Alphavirus are widely recognized for their potential to cause severe disease. Despite this recognition, there are no antiviral therapeutics, or safe and effective vaccines, currently available to treat alphaviral infection. Alphaviruses utilize the host cell machinery to efficiently establish and complete their life cycle. However, the extent and importance of host-pathogen RNA-protein interactions are woefully undercharacterized. The efforts detailed in this study fill this critical gap, and the significance of this research is 3-fold. First, the data presented here fundamentally expand the scope and understanding of alphavirus host-pathogen interactions. Second, this study identifies the sites of interaction for several prioritized interactions and defines the contribution of the RNA-protein interaction at the molecular level. Finally, these studies build a strategy by which the importance of the given host-pathogen interactions may be assessed in the future, using a mouse model of infection.

Dataset Information

IrCLIP platform for efficient characterization of protein-RNA interactions.

Publications

irCLIP platform for efficient characterization of protein-RNA interactions.

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