iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution

Abstract

The unique composition and spatial arrangement of RNA-binding proteins (RBPs) on a transcript guide the diverse aspects of posttranscriptional regulation 1 . Therefore, an essential step towards understanding transcript regulation at the molecular level is to gain positional information on the binding sites of RBPs 2 .Protein-RNA interactions can be studied using biochemical methods, but these approaches do not address RNA binding in its native cellular context. Initial attempts to study protein-RNA complexes in their cellular environment employed affinity purification or immunoprecipitation combined with differential display or microarray analysis (RIP-CHIP) [3][4][5] . These approaches were prone to identifying indirect or nonphysiological interactions 6 . In order to increase the specificity and positional resolution, a strategy referred to as CLIP (UV cross-linking and immunoprecipitation) was introduced 7,8 . CLIP combines UV cross-linking of proteins and RNA molecules with rigorous purification schemes including denaturing polyacrylamide gel electrophoresis. In combination with high-throughput sequencing technologies, CLIP has proven as a powerful tool to study protein-RNA interactions on a genome-wide scale (referred to as HITS-CLIP or CLIP-seq) 9,10 . Recently, PAR-CLIP was introduced that uses photoreactive ribonucleoside analogs for cross-linking 11,12 .Despite the high specificity of the obtained data, CLIP experiments often generate cDNA libraries of limited sequence complexity. This is partly due to the restricted amount of co-purified RNA and the two inefficient RNA ligation reactions required for library preparation. In addition, primer extension assays indicated that many cDNAs truncate prematurely at the crosslinked nucleotide 13 . Such truncated cDNAs are lost during the standard CLIP library preparation protocol. We recently developed iCLIP (individual-nucleotide resolution CLIP), which captures the truncated cDNAs by replacing one of the inefficient intermolecular RNA ligation steps with a more efficient intramolecular cDNA circularization (Figure 1) 14 . Importantly, sequencing the truncated cDNAs provides insights into the position of the cross-link site at nucleotide resolution. We successfully applied iCLIP to study hnRNP C particle organization on a genome-wide scale and assess its role in splicing regulation 14 .

Severe muscle wasting and denervation in mice lacking the RNA-binding protein ZFP106

“…5A) (30). To detect ZFP106-RNA complexes, cells transfected with ZFP106 fusions were cross-linked with UV light and immunoprecipitated using an anti-TY1 antibody.…”

Interleukin 1β Regulation of the System xc− Substrate-specific Subunit, xCT, in Primary Mouse Astrocytes Involves the RNA-binding Protein HuR

“…RNA Immunoprecipitation-Astrocytes were treated with recombinant murine IL-1␤ (3-5 ng/ml; R&D Systems, Minneapolis, MN) in an incubation/vehicle buffer of media stock (see above) containing 0.1% fatty-acid free BSA (Sigma) for 4 h. The medium was aspirated, 6 ml of ice-cold PBS was added, and the cells irradiated to 150 mJ/cm 2 (Stratagene; UV Stratalinker 1800) (46,47). Cells harvested by scraping were collected and spun (600 ϫ g, 5 min, 4°C).…”

The increasing diversity of functions attributed to the SAFB family of RNA-/DNA-binding proteins

“…Since SAFB1 is localised to perichromatin fibrils and is known to bind mRNA, it seemed likely that at least some of the SAFB is bound to newly transcribed pre-mRNA and involved in mRNA processing. Because target RNA sequences had not previously been identified for SAFB proteins, we used individual nucleotide resolution cross-linking and immunoprecipitation (iCLIP) with deep sequencing [48,49] to identify binding sites and determine which RNA species are bound by SAFB1.…”