<i>In vivo</i>discovery of RNA proximal proteins in human cells via proximity-dependent biotinylation

Lin, Xianzhi; Fonseca, Marcos A. S.; Corona, Rosario I.; Lawrenson, Kate

doi:10.1101/2020.02.28.970442

Cited by 7 publications

(5 citation statements)

References 142 publications

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“…RNA-centric PL methods (RapID; Ramanathan et al, 2018 ), RBPL ( Lu and Wei, 2019 ), RNA-BioID ( Mukherjee et al, 2019 ; Han et al, 2020 ), CARPID ( Yi et al, 2020 ), CBRPP ( Li et al, 2021b ), CRUIS ( Zhang et al, 2020b ), and RPL ( Lin et al, 2020 ) can be used to study dynamic interactomes of different RNA species and to identify regulators of RNA activity, localization, and stability. For targeting, they employ either stem loops, which are added to the RNA of interest and bound by the corresponding viral coat proteins or dCas13 and sequence-specific guide RNAs ( Figure 2 ).…”

Section: Oh the Possibilities!—applications For Enzymatic Plmentioning

confidence: 99%

Advances in enzyme-mediated proximity labeling and its potential for plant research

Mair

Bergmann

2021

Plant Physiology

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Cellular processes rely on the intimate interplay of different molecules, including DNA, RNA, proteins and metabolites. Obtaining and integrating data on their abundance and dynamics at high temporal and spatial resolution is essential for our understanding of plant growth and development. In the past decade, enzymatic proximity labeling (PL) has emerged as a powerful tool to study local protein and nucleotide ensembles, discover protein-protein and -nucleotide interactions and resolve questions about protein localization and membrane topology. An ever-growing number and continuous improvement of enzymes and methods keeps broadening the spectrum of possible applications for PL and makes it more accessible to different organisms, including plants. While initial PL experiments in plants required high expression levels and long labeling times, recently developed faster enzymes now enable PL of proteins on a cell type-specific level, even with low-abundant baits, and in different plant species. Moreover, expanding the use of PL for additional purposes, such as identification of locus-specific gene regulators or high-resolution electron microscopy may now be in reach. In this review, we give an overview of currently available PL enzymes and their applications in mammalian cell culture and plants. We discuss challenges and limitations of PL methods and highlight open questions and possible future directions for PL in plants.

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Section: Oh the Possibilities!—applications For Enzymatic Plmentioning

confidence: 99%

Advances in enzyme-mediated proximity labeling and its potential for plant research

Mair

Bergmann

2021

Plant Physiology

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“…Proximity Proteome of Specific RNA Transcripts: CRISPR-Based Approaches The discovery of the CRISPR-Cas13 RNA targeting system (Box 1) provides new opportunities to recruit proximity-labeling enzymes to an RNA transcript of interest. This new methodology has been recently applied by several groups [56,[58][59][60][61]. Zhang and colleagues developed a Box 1.…”

Section: Open Accessmentioning

confidence: 99%

RNA-Centric Methods: Toward the Interactome of Specific RNA Transcripts

Gräwe

Stelloo

Hout

et al. 2021

Trends in Biotechnology

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RNA-protein interactions play an important role in numerous cellular processes in health and disease. In recent years, the global RNA-bound proteome has been extensively studied, uncovering many previously unknown RNA-binding proteins. However, little is known about which particular proteins bind to which specific RNA transcript. In this review, we provide an overview of methods to identify RNA-protein interactions, with a particular focus on strategies that provide insights into the interactome of specific RNA transcripts. Finally, we discuss challenges and future directions, including the potential of CRISPR-RNA targeting systems to investigate endogenous RNA-protein interactions. Importance of Studying RNA-Protein ComplexesThe various types of RNA, such as mRNAs, rRNAs, and long noncoding (lnc)RNAs, are involved in a multitude of cellular processes. The classical view of RNAs serving merely as a template for protein synthesis has been expanded to catalytic, structural, and regulatory functions [1,2]. RNAs interact with RNA-binding proteins (RBPs; see Glossary), which play an essential role in RNA fate and function, such as in mRNA processing and translation [3]. In addition, RNA binding can also change the fate and function of proteins, for example by regulating protein stability and localization [4,5]. The importance of these interactions also becomes clear in the context of diseases that are characterized by perturbed RNA-protein interactions. Any alteration in expression level, structure, localization, or modification of either RNA or protein may disrupt these interactions and result in deregulation of the involved cellular processes [6,7]. For example, the RNA modification N6-methyladenosine (m6A) affects protein binding, and alterations in m6A levels are frequently observed in cancer [8][9][10].To characterize RNA-protein interactions, a rapidly expanding toolbox of methods is available, often classified into protein-centric and RNA-centric methods. Protein-centric methods, such as crosslinking immunoprecipitation (CLIP), rely on immunoprecipitation of the protein of interest followed by sequencing of the associated RNAs [11,12]. Conversely, RNA-centric methods rely on isolation of proteins associated with the RNA of interest. These methods led to the identification of many RBPs, including unexpected proteins, such as metabolic enzymes and transcription factors (reviewed in [5]). In this review, we provide an overview of the current and emerging RNAcentric approaches (Figure 1). In addition, we highlight the advantages, disadvantages, and challenges that have to be overcome to improve current methods. Furthermore, we discuss potential technical innovations to provide robust insights into endogenous protein-RNA interactomes. Global RNA-Binding ProteomeTo gain insight into the function of RNA-protein interactions, several methods have been developed in recent years to identify the global RNA-bound proteome. These methods preserve cellular RNA-protein interactions by crosslinking, which is then followed by c...

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“…dCas13 can be fused to GFP, enabling imaging of RNA [ 94 ], to ADAR2, enabling editing of RNA [ 108 ] or to labeling enzymes like APEX2 [ 95 , 97 ] to probe for interacting proteins. A number of Cas13 variants have been used as guiding proteins, e.g., RfxCas13 [ 93 ], PspCas13b [ 97 ], CasRx [ 96 ], and LwaCas13a [ 95 ]. Tethering of the fusion protein to RNA can be improved by using a gRNA array instead of single gRNAs [ 96 ].…”

Section: Proximity Labeling Of Rna–protein Interactions: Finding the Protein Partnersmentioning

confidence: 99%

“…Multiple gRNAs are also used in a strategy aimed at reducing the number of false positive interactors. Lin et al targeted a dPspCas13b-APEX2 fusion to the U1 snRNA using three gRNAs [ 97 ]. Each of the gRNAs, binding to a different single-stranded RNA region, was expressed in a separate cell line but the interactome data obtained with each gRNA were aligned.…”

Section: Proximity Labeling Of Rna–protein Interactions: Finding the Protein Partnersmentioning

confidence: 99%

“…Special care has also to be taken in the design of the fusion construct of dCas13 and the biotinylation enzyme, as it seems important to insert a linker between both parts to uncouple the activities of the two [ 93 ]. Additionally, an optimal molar ratio between the fusion construct and the gRNA [ 97 ] as well as a stable genomic integration of the fusion protein construct and the control of its expression via an inducible promoter (e.g., via the tet-on system) was reported to be essential for a good signal-to-noise ratio in labeling [ 93 , 95 ].…”

Section: Proximity Labeling Of Rna–protein Interactions: Finding the Protein Partnersmentioning

confidence: 99%

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RNA Proximity Labeling: A New Detection Tool for RNA–Protein Interactions

et al. 2021

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Multiple cellular functions are controlled by the interaction of RNAs and proteins. Together with the RNAs they control, RNA interacting proteins form RNA protein complexes, which are considered to serve as the true regulatory units for post-transcriptional gene expression. To understand how RNAs are modified, transported, and regulated therefore requires specific knowledge of their interaction partners. To this end, multiple techniques have been developed to characterize the interaction between RNAs and proteins. In this review, we briefly summarize the common methods to study RNA–protein interaction including crosslinking and immunoprecipitation (CLIP), and aptamer- or antisense oligonucleotide-based RNA affinity purification. Following this, we focus on in vivo proximity labeling to study RNA–protein interactions. In proximity labeling, a labeling enzyme like ascorbate peroxidase or biotin ligase is targeted to specific RNAs, RNA-binding proteins, or even cellular compartments and uses biotin to label the proteins and RNAs in its vicinity. The tagged molecules are then enriched and analyzed by mass spectrometry or RNA-Seq. We highlight the latest studies that exemplify the strength of this approach for the characterization of RNA protein complexes and distribution of RNAs in vivo.

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In vivodiscovery of RNA proximal proteins in human cells via proximity-dependent biotinylation

Cited by 7 publications

References 142 publications

Advances in enzyme-mediated proximity labeling and its potential for plant research

Advances in enzyme-mediated proximity labeling and its potential for plant research

RNA-Centric Methods: Toward the Interactome of Specific RNA Transcripts

RNA Proximity Labeling: A New Detection Tool for RNA–Protein Interactions

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