Advances and challenges in the detection of transcriptome‐wide protein–RNA interactions

RNA binding proteins (RBPs) play key roles in determining cellular behavior by manipulating the processing of target RNAs. Robust methods are required to detect the numerous binding sites of RBPs across the transcriptome. RNA‐immunoprecipitation followed by sequencing (RIP‐seq) and crosslinking foll...

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Published in:Wiley interdisciplinary reviews. RNA 2018-01, Vol.9 (1), p.e1436-n/a
Main Authors: Wheeler, Emily C., Van Nostrand, Eric L., Yeo, Gene W.
Format: Article
Language:English
Subjects:
Binding sites
Gene expression
Immunoprecipitation
Opinion
Peptide mapping
Polymerase chain reaction
Proteins
Protein–RNA Interactions: Functional Implications
Protein–RNA Recognition
Radioactive labeling
RNA Analyses in Cells
RNA processing
RNA-binding protein
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Summary:RNA binding proteins (RBPs) play key roles in determining cellular behavior by manipulating the processing of target RNAs. Robust methods are required to detect the numerous binding sites of RBPs across the transcriptome. RNA‐immunoprecipitation followed by sequencing (RIP‐seq) and crosslinking followed by immunoprecipitation and sequencing (CLIP‐seq) are state‐of‐the‐art methods used to identify the RNA targets and specific binding sites of RBPs. Historically, CLIP methods have been confounded with challenges such as the requirement for tens of millions of cells per experiment, low RNA yields resulting in libraries that contain a high number of polymerase chain reaction duplicated reads, and technical inconveniences such as radioactive labeling of RNAs. However, recent improvements in the recovery of bound RNAs and the efficiency of converting isolated RNAs into a library for sequencing have enhanced our ability to perform the experiment at scale, from less starting material than has previously been possible, and resulting in high quality datasets for the confident identification of protein binding sites. These, along with additional improvements to protein capture, removal of nonspecific signals, and methods to isolate noncanonical RBP targets have revolutionized the study of RNA processing regulation, and reveal a promising future for mapping the human protein‐RNA regulatory network. WIREs RNA 2018, 9:e1436. doi: 10.1002/wrna.1436 This article is categorized under: RNA Interactions with Proteins and Other Molecules > Protein–RNA Recognition RNA Interactions with Proteins and Other Molecules > Protein–RNA Interactions: Functional Implications RNA Methods > RNA Analyses in Cells Methods to capture protein‐RNA interactions. Different techniques are required to capture single‐stranded (green), double‐stranded (blue), and indirect (yellow) RNA interactions. Crosses (X) in red mark RNA sites that are crosslinked to the RNA binding protein. (right) UV treatment at 254 nm preferentially captures binding in single‐stranded regions. (bottom right) 0.1% formaldehyde treatment captures all protein‐protein and protein‐RNA interactions. (bottom left) RNA immunoprecipitation (RIP) uses a native pulldown (no crosslinking) to capture binding events with antibody selection. Optimized RNA digestion conditions can reveal specific binding sites with RIP. (left) Photoactivatable ribonucleoside analog treatment (PAR) increases UV crosslinking efficiency at 365 nm. (top left)
ISSN:1757-7004
1757-7012
DOI:10.1002/wrna.1436