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

Abstract

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.1436This article is categorized under: RNA Interactions with Proteins and Other Molecules > Protein–RNA RecognitionRNA Interactions with Proteins and Other Molecules > Protein–RNA Interactions: Functional ImplicationsRNA Methods > RNA Analyses in Cells

A Genetically Encoded Diazirine Analogue for RNA–Protein Photo‐crosslinking

“…Therefore, UV crosslinking stalls interaction dynamics and covalently links the interacting partners together in a way that withstands stringent denaturing downstream enrichment and analysis steps. The introduction of UV crosslinking has revolutionized the field and boosted high‐throughput studies of RPBs and RNA‐protein interactions . At the same time, conventional UV crosslinking possesses a range of intrinsic drawbacks which limit the performance of the experimental procedures .…”

“…The introduction of UV crosslinking has revolutionized the field and boosted high‐throughput studies of RPBs and RNA‐protein interactions . At the same time, conventional UV crosslinking possesses a range of intrinsic drawbacks which limit the performance of the experimental procedures . For instance, the chemical yield of the photo‐crosslinking step is typically very low (<1 %).…”

“…Also, 254 nm UV light produces undesirable intra‐ and interstrand RNA‐RNA crosslinks and phosphodiester backbone breaks . Moreover, 254 nm UV crosslinking has an inherent bias coming from the unequal photochemical reactivity of different nucleobases toward different amino acid side chains and also from different reactivity of structured and unstructured stretches of RNA . The mentioned factors complicate both the processing of crosslinked samples and interpretation of the results.…”

Empowering the annotation and discovery of structured RNAs with scalable and accessible integrative clustering

“…Firstly, eCLIP is an improved protocol with a size-matched input to capture background RNAs of the CLIP protocol [62]. On the other hand, PAR-CLIP is known to have relatively higher false positive rates [87]. Secondly, the ROQ domain of Roquin-1 has two RNA binding sites, one that specifically recognizes CDE-like stem-loops and one that binds to double-stranded RNAs [86,88].…”

Systematic Analysis of Targets of Pumilio (PUM)-Mediated mRNA Decay Identifies a Role of PUM1 in Regulating DNA Damage Response Pathway

“…Hence, RBPs play a central role in gene regulation by providing specificity to mRNA degradation. Studies that applied high-throughput sequencing methods, such as RIP-seq or CLIP-seq [8][9][10] , revealed that an individual RBP can bind to 100 to 1,000 mRNAs. For the RBPs involved in mRNA decay, not all mRNAs that bind the RBP are targets of degradation.…”