RNA transcripts are never alone in the cell. In particular, RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes bind mRNAs in a sequence-dependent manner and have an important role in regulating gene expression post-transcriptionally. Hundreds of these RBPs and microRNA-containing ribonucleoprotein complexes modulate the maturation, transport, editing and translation of mRNAs in the cell, but techniques for precise identification of where these molecular complexes are bound to the mRNAs in vivo are still lacking.

UV-light irradiation is known to covalently cross-link RNA to RNA-bound protein complexes in living cells. In an approach called cross-linking and immunoprecipitation (CLIP), live cells are exposed to UV light to freeze the RNA-protein interactions and allow isolation of proteins of interest together with their associated RNAs by immunoprecipitation. High-throughput sequencing of the reverse-transcribed, cross-linked RNA results in a set of sequence reads representing RNA sequences that were bound to the particular RNA-binding complex in the cell.

But this approach has some limitations. Photo-cross-linking with 254-nm UV light, as is done in CLIP, is a relatively inefficient process that leads to the difficult problem of separating signal from background. Biochemical separation of cross-linked from non-cross-linked RNA is a demanding process and still requires the use of material from gene knockout organisms, computational models or filtering of expected sites or motifs to combat the inherent noise.

To solve these problems comes the next generation of high-throughput RNA cross-linking, photoactivatable ribonucleoside–enhanced CLIP (PAR-CLIP), developed by Thomas Tuschl's group at the Rockefeller University in collaboration with the group of Mihaela Zavolan at the Biozentrum in the University of Basel. PAR-CLIP introduces the use of photoactivatable nucleosides as efficient and nontoxic cross-linkers that are well incorporated into the RNA. In this method, cells are grown in the presence of 4-thiouridine (4SU) and 4SU-substituted RNA is cross-linked using 365-nm UV light, which is safer and more efficient than cross-linking unsubstituted RNA using 254-nm UV light.

The true advance of this technique arose from a fortuitous finding: the cross-linked nucleosides cause a specific base change during reverse transcription that leaves a permanent mark in the place where the protein complex originally stood on the RNA. As the reverse transcriptase misincorporates guanine (G) opposite the cross-linked 4SU base, scoring for thymidine (T) to cytidine (C) transitions in the sequenced cDNA opens the door to precisely mapping the binding sites for RNA-binding proteins, and it allows the separation of bona fide cross-linked RNA sequences from noise.

Tuschl and co-workers performed a herculean demonstration of the powers of PAR-CLIP with transcriptome-wide mapping of binding sites for 13 RBPs, of which seven proteins are involved in microRNA targeting. By focusing on sequences that bear the hallmark T to C transition, the authors defined binding motifs for RBPs such as insulin-like growth factor-2 and identified previously unknown targets for microRNAs.

These data sets verify the robustness of the technique by confirming known binding partners, and also provide a wealth of new RNA-interaction maps. “The struggle is now to prioritize all this data-mining,” says Tuschl. “We want to know if there are genetic variations in these binding sites in humans and whether some of them contribute to genetic diseases that implicate loss of an RNA-binding protein, such as fragile X mental retardation or familial amyotrophic lateral sclerosis.”

The future of PAR-CLIP is bright, promising new and imaginative possibilities for experimentation that will bring science a step closer to unraveling the mysteries of gene regulation.