The unique composition and spatial arrangement of RNA-binding proteins (RBPs) on 
a transcript guide the diverse aspects of post-transcriptional regulation. 
Therefore, an essential step towards understanding transcript regulation at the 
molecular level is to gain positional information on the binding sites of RBPs. 
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). These approaches were prone to identifying 
indirect or non-physiological interactions. In order to increase the specificity 
and positional resolution, a strategy referred to as CLIP (UV cross-linking and 
immunoprecipitation) was introduced. 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). 
Recently, PAR-CLIP was introduced that uses photoreactive ribonucleoside analogs 
for cross-linking. 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. 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). 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.