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In-cell identification and measurement of RNA-protein interactions.

ABSTRACT: Regulatory RNAs exert their cellular functions through RNA-binding proteins (RBPs). Identifying RNA-protein interactions is therefore key for a molecular understanding of regulatory RNAs. To date, RNA-bound proteins have been identified primarily through RNA purification followed by mass spectrometry. Here, we develop incPRINT (in cell protein-RNA interaction), a high-throughput method to identify in-cell RNA-protein interactions revealed by quantifiable luminescence. Applying incPRINT to long noncoding RNAs (lncRNAs), we identify RBPs specifically interacting with the lncRNA Firre and three functionally distinct regions of the lncRNA Xist. incPRINT confirms previously known lncRNA-protein interactions and identifies additional interactions that had evaded detection with other approaches. Importantly, the majority of the incPRINT-defined interactions are specific to individual functional regions of the large Xist transcript. Thus, we present an RNA-centric method that enables reliable identification of RNA-region-specific RBPs and is applicable to any RNA of interest.

SUBMITTER: Graindorge A

PROVIDER: S-EPMC6876571 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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In-cell identification and measurement of RNA-protein interactions.

Graindorge Antoine A Pinheiro Inês I Nawrocka Anna A Mallory Allison C AC Tsvetkov Peter P Gil Noa N Carolis Carlo C Buchholz Frank F Ulitsky Igor I Heard Edith E Taipale Mikko M Shkumatava Alena A

Nature communications 20191122 1

Regulatory RNAs exert their cellular functions through RNA-binding proteins (RBPs). Identifying RNA-protein interactions is therefore key for a molecular understanding of regulatory RNAs. To date, RNA-bound proteins have been identified primarily through RNA purification followed by mass spectrometry. Here, we develop incPRINT (in cell protein-RNA interaction), a high-throughput method to identify in-cell RNA-protein interactions revealed by quantifiable luminescence. Applying incPRINT to long n ...[more]

PMID: 31757954

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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

In-cell identification and measurement of RNA-protein interactions.

Publications

In-cell identification and measurement of RNA-protein interactions.

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