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

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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.

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 (PAR) analog treatment increases UV crosslinking efficiency at 365 nm. Top left: Methylene blue intercalates between the bases of double‐stranded RNA to allow crosslinking in double‐stranded regions in the presence of visible light. Top right: Protein–RNA interaction sites are marked by exogenous RNA modifications. This requires creating a fusion protein to modify RNA near binding sites with biotinylation (BioTag‐BirA) or A‐to‐I RNA editing (ADAR).
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Quantification of background signal with size‐matched input (SM‐Input). The 2% of lysate is taken prior to IP as the input sample. ’Sticky’ RBPs (yellow) are not completely purified away and contaminate the IP sample. The input and IP are run in parallel on the protein gel and extracted from the nitrocellulose membrane at the same size range. Called peaks are then normalized by dividing the number of reads in the IP by the number of reads in the input to remove signal coming from background RNAs (yellow). The enrichment score is a rank‐based metric for specificity of binding.
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Comparison of CLIP‐seq library preparation protocols. HITS‐CLIP/CLIP‐seq perform adapter ligation on both ends of the RNA while other protocols only ligate an RNA adapter on the 3′ end. RNA digested fragments are visualized with radiolabeling (HITS‐CLIP/CLIP‐seq and iCLIP), infrared dye imaging (irCLIP), or not at all (eCLIP). RNA is then transferred and isolated from a nitrocellulose membrane. In the case of eCLIP, a size‐matched input sample is excised to control for background. For all, reverse transcription (RT) generates cDNA using the 3′RNA adapter as the priming site. As the RT enzyme commonly terminates at crosslinking sites, there will be a mixture of full length and truncated fragments. iCLIP and irCLIP have a circularization step to put the second adapter on the DNA fragment. Ligation in irCLIP has been highly optimized to improve efficiency. eCLIP uses a second ligation step that has also been optimized for efficiency. HITS‐CLIP/CLIP‐seq fragments that have incomplete RT cannot PCR amplify due to the loss of the second adapter sequence. For all, cDNA fragments are then PCR amplified to generate enough material for sequencing. Recent methods (irCLIP, eCLIP) have routinely high complexity libraries generated from a low number of PCR cycles.
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RNA Interactions with Proteins and Other Molecules > Protein–RNA Interactions: Functional Implications
RNA Interactions with Proteins and Other Molecules > Protein–RNA Recognition
RNA Methods > RNA Analyses in Cells

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