Methods to study RNA–protein interactions

Ramanathan, Muthukumar; Porter, Douglas F.; Khavari, Paul A.

doi:10.1038/s41592-019-0330-1

Cited by 273 publications

(258 citation statements)

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“…It provides direct visualization of the success of library preparation steps, allows multiplexing based on two adapters, and determines ligation efficiency. A major limitation to this approach is its reliance on UV cross-linking as a proxy for in vivo interactions 20 .…”

Section: Discussionmentioning

confidence: 99%

easyCLIP Quantifies RNA-Protein Interactions and Characterizes Recurrent PCBP1 Mutations in Cancer

Porter

Khavari

2019

Preprint

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35RNA-protein interactions mediate a host of cellular processes, underscoring the need 36 for methods to quantify their occurrence in living cells. RNA interaction frequencies 37 for the average cellular protein are undefined, however, and there is no quantitative 38 threshold to define a protein as an RNA-binding protein (RBP). Ultraviolet (UV) cross-39 linking immunoprecipitation (CLIP)-sequencing, an effective and widely used 40 means of characterizing RNA-protein interactions, would particularly benefit from 41 the capacity to quantitate the number of RNA cross-links per protein per cell. In 42 addition, CLIP-seq methods are difficult, have high experimental failure rates and 43 many ambiguous analytical decisions. To address these issues, the easyCLIP 44 method was developed and used to quantify RNA-protein interactions for a panel 45of known RBPs as well as a spectrum of random non-RBP proteins. easyCLIP 46 provides the advantages of good efficiency compared to current standards, a 47 simple protocol with a very low failure rate, troubleshooting information that 48 includes direct visualization of prepared libraries without amplification, and a new 49 form of analysis. easyCLIP, which uses sequential on-bead ligation of 5' and 3' 50 adapters tagged with different infrared dyes, classified non-RBPs as those with a 51 per protein RNA cross-link rate of <0.1%, with most RBPs substantially above this 52 threshold, including Rbfox1 (18%), hnRNPC (22%), CELF1 (11%), FBL (2%), and 53 STAU1 (1%). easyCLIP with the PCBP1 L100 RBP mutant recurrently seen in cancer 54 quantified increased RNA binding compared to wild-type PCBP1 and suggested a 55 potential mechanism for this RBP mutant in cancer. easyCLIP provides a simple, 56 efficient and robust method to both obtain both traditional CLIP-seq information 57and to define actual RNA interaction frequencies for a given protein, enabling 58 quantitative cross-RBP comparisons as well as insight into RBP mechanisms. 59 60 61 62 65challenges of integrating such data between RNA-binding proteins (RBPs) was recently 66 highlighted 1 . The physical reality of RNA-protein interactions is their individual occurrence in 67individual cells, which may be abstracted to an average complex number per-cell in a 68population. The RNA-protein complex count per-cell may be normalized to derive the number 69 of complexes per-interaction partner. It is these frequencies, per-cell and per-interaction 70 partner, that are the most basic characterizations of RNA-protein interaction networks. 71Determining the targets of an RBP by enrichment over negative control immunopurifications, 72or by clustering of cross-links, or many such other approaches, are all ultimately inferring that 73the absolute count of an RNA-protein complex in the cell is abnormally high. The estimation 74of per-cell and per-protein absolute quantities provide the ultimate framework for describing 75 a global and widely reproducible view of RNA-protein interactions. 76 77There is currently no general method to estimate abs...

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Section: Discussionmentioning

confidence: 99%

easyCLIP Quantifies RNA-Protein Interactions and Characterizes Recurrent PCBP1 Mutations in Cancer

Porter

Khavari

2019

Preprint

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“…The most commonly used method to identify RNAs that are directly bound by a specific protein is CLIP (Cross Linking and Immunoprecipitation) (Chakrabarti, Haberman, Praznik, Luscombe, & Ule, ; Darnell, ; Hannigan, Zagore, & Licatalosi, ; F. C. Y. Lee & Ule, ; Ramanathan, Porter, & Khavari, ; Ule, Hwang, & Darnell, ). CLIP methods typically use an antibody to immunoprecipitate (IP) native or epitope‐tagged proteins that have been UV‐crosslinked to RNA, or affinity precipitation of an affinity‐tagged protein (Lapointe et al, ; McMahon et al, ; Wu et al, ).…”

Section: Approaches To Measure the Dynamics Of Rna–protein Interactiomentioning

confidence: 99%

Approaches for measuring the dynamics of RNA–protein interactions

Licatalosi

Jankowsky

2019

WIREs RNA

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RNA-protein interactions are pivotal for the regulation of gene expression from bacteria to human. RNA-protein interactions are dynamic; they change over biologically relevant timescales. Understanding the regulation of gene expression at the RNA level therefore requires knowledge of the dynamics of RNA-protein interactions. Here, we discuss the main experimental approaches to measure dynamic aspects of RNA-protein interactions. We cover techniques that assess dynamics of cellular RNA-protein interactions that accompany biological processes over timescales of hours or longer and techniques measuring the kinetic dynamics of RNA-protein interactions in vitro.

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“…However, CLIP suffers from strong experimental biases 8 , is limited in binding-site resolution, assesses only a single protein at a time, and requires a unique antibody or exogenous tag. Methods that focus on cataloging RNA-binding proteins, like mass spectrometry, cannot simultaneously locate protein-binding sites on RNA nor easily prioritize proteins in terms of function 9 . These limitations obscure the importance of binding by multiple proteins to individual RNAs.…”

Section: Introductionmentioning

confidence: 99%

RNP-MaP: In-cell analysis of protein interaction networks defines functional hubs in RNA

Weidmann¹,

Mustoe²,

Jariwala³

et al. 2020

Preprint

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RNAs interact with networks of proteins to form complexes (RNPs) that govern many biological processes, but these networks are currently impossible to examine in a comprehensive way.We developed a live-cell chemical probing strategy for mapping protein interaction networks in any RNA with single-nucleotide resolution. This RNP-MaP strategy (RNP network analysis by mutational profiling) simultaneously detects binding by and cooperative interactions involving multiple proteins with single RNA molecules. RNP-MaP revealed that two structurally related, but sequence-divergent noncoding RNAs, RNase P and RMRP, share nearly identical RNP networks and, further, that protein interaction network hubs identify function-critical sites in these RNAs. RNP-MaP identified numerous protein interaction networks within the XIST long noncoding RNA that are conserved between mouse and human RNAs and distinguished communities of proteins that network together on XIST. RNP-MaP data show that the Xist E region is densely networked by protein interactions and that PTBP1, MATR3, and TIA1 proteins each interface with the XIST E region via two distinct interaction modes; and we find that the XIST E region is sufficient to mediate RNA foci formation in cells. RNP-MaP will enable discovery and mechanistic analysis of protein interaction networks across any RNA in cells. RESULTS RNP-MaP ValidationTo comprehensively map protein interaction networks of an RNA of interest, we identified a cellpermeable reagent, NHS-diazirine (SDA), that can rapidly label RNA nucleotides at sites of protein binding. SDA has two reactive moieties: a succinimidyl ester and a diazirine ( Fig. 1a).Succinimidyl esters react to form amide bonds with amines such as those found in lysine side chains. When photoactivated with long-wavelength ultraviolet light (UV-A, 365 nm), diazirines form carbene or diazo intermediates 10 , which are broadly reactive to nucleotide sugar and base moieties. The two-step reaction of SDA thus crosslinks protein lysine residues with RNA with a distance governed by SDA linker length (4 Å) and lysine flexibility (6 Å). Lysine is one of the most prevalent amino acids in RNA-binding domains 11 , and photo-intermediate lifetimes are short; thus, SDA is expected to crosslink short-range RNA-protein interactions relatively independently of local RNA structure or protein properties. To perform crosslinking, live cells are treated with SDA for a short time and then exposed to UV-A light.To detect SDA-mediated RNA-protein crosslinks, we use the MaP reverse transcription technology ( Fig. 1b). SDA-treated cells are lysed and crosslinked proteins are digested to short peptide adducts. Adduct-containing RNA is then used as a template for relaxed-fidelity reverse transcription 12,13 . Under MaP conditions, reverse transcriptase reads through adduct-containing nucleotides and incorporates non-templated nucleotides into the product DNA at the site of RNAprotein crosslinks. Importantly, because transcription reads through adducts, RNP-MaP detects multiple pro...

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Methods to study RNA–protein interactions

Cited by 273 publications

References 103 publications

easyCLIP Quantifies RNA-Protein Interactions and Characterizes Recurrent PCBP1 Mutations in Cancer

easyCLIP Quantifies RNA-Protein Interactions and Characterizes Recurrent PCBP1 Mutations in Cancer

Approaches for measuring the dynamics of RNA–protein interactions

RNP-MaP: In-cell analysis of protein interaction networks defines functional hubs in RNA

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