RNA transcripts are subjected to post-transcriptional gene regulation by 
interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing 
ribonucleoprotein complexes (miRNPs) that are often expressed in a cell-type 
dependently. To understand how the interplay of these RNA-binding factors 
affects the regulation of individual transcripts, high resolution maps of in 
vivo protein-RNA interactions are necessary. A combination of genetic, 
biochemical and computational approaches are typically applied to identify 
RNA-RBP or RNA-RNP interactions. Microarray profiling of RNAs associated with 
immunopurified RBPs (RIP-Chip) defines targets at a transcriptome level, but its 
application is limited to the characterization of kinetically stable 
interactions and only in rare cases allows to identify the RBP recognition 
element (RRE) within the long target RNA. More direct RBP target site 
information is obtained by combining in vivo UV crosslinking with 
immunoprecipitation followed by the isolation of crosslinked RNA segments and 
cDNA sequencing (CLIP). CLIP was used to identify targets of a number of RBPs. 
However, CLIP is limited by the low efficiency of UV 254 nm RNA-protein 
crosslinking, and the location of the crosslink is not readily identifiable 
within the sequenced crosslinked fragments, making it difficult to separate 
UV-crosslinked target RNA segments from background non-crosslinked RNA fragments 
also present in the sample. We developed a powerful cell-based crosslinking 
approach to determine at high resolution and transcriptome-wide the binding 
sites of cellular RBPs and miRNPs that we term PAR-CliP 
(Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) 
(see Fig. 1A for an outline of the method). The method relies on the 
incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine 
(4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. 
Irradiation of the cells by UV light of 365 nm induces efficient crosslinking of 
photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. 
Immunoprecipitation of the RBP of interest is followed by isolation of the 
crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a 
cDNA library and deep sequenced using Solexa technology. One characteristic 
feature of cDNA libraries prepared by PAR-CliP is that the precise position of 
crosslinking can be identified by mutations residing in the sequenced cDNA. When 
using 4-SU, crosslinked sequences thymidine to cytidine transition, whereas 
using 6-SG results in guanosine to adenosine mutations. The presence of the 
mutations in crosslinked sequences makes it possible to separate them from the 
background of sequences derived from abundant cellular RNAs. Application of the 
method to a number of diverse RNA binding proteins was reported in Hafner et al.