RNA–protein interaction detection in living cells

Ramanathan, Muthukumar; Majzoub, Karim; Rao, Deepti; Neela, Poornima H.; Zarnegar, Brian; Mondal, Smarajit; Roth, Julien G.; Gai, Hongwei; Kovalski, Joanna; Siprashvili, Zurab; Palmer, Theo D.; Carette, Jan E.; Khavari, Paul A.

doi:10.1038/nmeth.4601

Cited by 241 publications

(273 citation statements)

References 45 publications

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“…Novel or improved applications using BioID ligase have spurred numerous follow-up articles including a smaller version of BioID with improved sensitivity and localization (22), its use for identifying protein-RNA interactions (23), split-BioID studies (24,25), and faster versions of BioID (26). Several conventional approaches have been utilized to stably introduce BioID-fusion proteins to cells including transfection (7), viral infection (27), and more recently, CRISPR-Cas9 (28).…”

Section: Introductionmentioning

confidence: 99%

BioID as a Tool for Protein-Proximity Labeling in Living Cells

Sears

May

Roux

2019

Methods in Molecular Biology

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BioID has become an increasingly utilized tool for identifying candidate protein-protein interactions (PPIs) in living cells. This method utilizes a promiscuous biotin ligase, called BioID, fused to a protein-of-interest that when expressed in cells can be induced to biotinylate interacting and proximate proteins over a period of hours, thus generating a history of protein associations. These biotinylated proteins are subsequently purified and identified via mass spectrometry. Compared to other conventional methods typically used to screen strong PPIs, BioID allows for the detection of weak and transient interactions within a relevant biological setting over a defined period of time. Here we briefly review the scientific progress enabled by the BioID technology, detail an updated protocol for applying the method to proteins in living cells, and offer insights for troubleshooting commonly encountered setbacks.

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Section: Introductionmentioning

confidence: 99%

BioID as a Tool for Protein-Proximity Labeling in Living Cells

Sears

May

Roux

2019

Methods in Molecular Biology

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“…By adapting the RNA antisense purification (RAP) method to purify a specific lncRNA complexes and identifying the interacting proteins using quantitative mass spectrometry, RAP-MS (RNA antisense purification by mass spectrometry) enables characterization of interacting proteins of a given lncRNA [37] (Table 2). RaPID (RNA purification and identification) allows for the isolation of specific mRNAs of interest and subsequent analysis of the associated proteins using mass spectrometry [38] (Table 2). A couple of methods have been developed to determine RNA-protein binding specificities.…”

Section: Rna Interactions With Proteinmentioning

confidence: 99%

RNA: interactions drive functionalities

2019

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RNA is produced from the majority of human genomic sequences, although only a relatively small portion of these transcripts has known functions. Diverse RNA species interact with RNA, DNA, proteins, lipids, and metabolites to form intricate molecular networks. In this review, we attempt to delineate diverse RNA functions by interaction types between RNA and other macromolecules. Through such interactions RNAs participate in essentially every major molecular function and process, including information flow and storage, environment sensing, signal transduction, and gene regulation at transcriptional and posttranscriptional levels. Through such interactions, RNAs promote or inhibit diverse biological processes, and act as catalyzer or quencher to modulate the pace of these progresses. Alterations and personal variations of these interactions are mechanistically coupled with disease etiology and phenotypical variations for clinical use.Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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“…This includes the use of RNA-capture methods, in which one can selectively purify a target RNA using antisense capture probes, followed by MS profiling of associated proteins [144][145][146][147][148][149]. Interestingly, Ramanathan et al [150] have recently developed a method using BirA-mediated proximity labeling paired with a kN-BoxB targeting system to selectively biotinylate and purify proteins that associate with an RNA of interest in live cells. While these approaches are used more broadly to interrogate RNA-protein interactions, without a specific focus on RNA localization, they are crucial in helping to identify RPBs that are likely to be implicated in RNA maturation and trafficking.…”

Section: Trans-acting Rna Localization Regulatory Elementsmentioning

confidence: 99%

Biological functions, regulatory mechanisms, and disease relevance of RNA localization pathways

et al. 2018

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The asymmetric subcellular distribution of RNA molecules from their sites of transcription to specific compartments of the cell is an important aspect of post-transcriptional gene regulation. This involves the interplay of intrinsic cis-regulatory elements within the RNA molecules with trans-acting RNAbinding proteins and associated factors. Together, these interactions dictate the intracellular localization route of RNAs, whose downstream impacts have wide-ranging implications in cellular physiology. In this review, we examine the mechanisms underlying RNA localization and discuss their biological significance. We also review the growing body of evidence pointing to aberrant RNA localization pathways in the development and progression of diseases.Keywords: Cis-regulatory elements; RNA disease; RNA localization; RNA-binding proteinsThe asymmetric organization of cells is a pervasive feature that ensures the homeostasis of prokaryotic and eukaryotic organisms. This relies on the capacity of cells to organize their contents, including lipids, nucleic acids, and proteins, into specific subcellular structures and organelles. While much of this organization has been attributed to subcellular protein transport [1], a growing body of work indicates that the regulated localization of RNA molecules is also a key process observed across evolutionary timescales [2][3][4][5][6]. This form of post-transcriptional regulation where coding and noncoding RNA molecules actively associate with trans-acting partners, such as RNA-binding proteins (RBPs), serves to dictate both the function and intra-or extracellular destiny of the RNA. During RNA localization, cis-regulatory elements found within the RNA molecule, whether coding or noncoding, act as recognition sites for the recruitment of trans-acting RBPs, which dictate the intra-/extra-cellular fate of the RNA and, ultimately, its functional state. Over the years, the wide-ranging implications of RNA localization on cellular physiology have become apparent, affecting many aspects of cell organization and function. Indeed, RNA localization pathways have been linked to numerous important processes, including developmental patterning, cell polarity, spindle assembly, formation of nonmembranous compartments, localized translation, and cell motility [2][3][4][5][6].

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RNA–protein interaction detection in living cells

Cited by 241 publications

References 45 publications

BioID as a Tool for Protein-Proximity Labeling in Living Cells

BioID as a Tool for Protein-Proximity Labeling in Living Cells

RNA: interactions drive functionalities

Biological functions, regulatory mechanisms, and disease relevance of RNA localization pathways

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