Transcriptome-Wide Identification of Protein-Binding Sites on RNA by PAR-CLIP (Photoactivatable Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation)
AUTHORS
ABSTRACT
Post-transcriptional regulation (PTR) of messenger RNAs (mRNAs) plays important roles in diverse cellular processes (Ambros 2004; Halbeisen, Galgano et al. 2008). The fates of mRNAs are determined predominantly by their interactions with RNA-binding proteins (RBPs) and non- coding, guide-RNA-containing ribonucleoprotein complexes (RNPs). Taken together, they form mRNA-containing ribonucleoprotein complexes (mRNPs). The RBPs influence the structure and interactions of the RNAs and play critical roles in their biogenesis, stability, function, transport and cellular localization (Moore 2005; Keene 2007; Glisovic, Bachorik et al. 2008).
Given that hundreds of RBPs and RNPs and their networks remain to be studied and evaluated in a cell-type-dependent manner, the development of powerful tools to determine their binding sites or RNA recognition elements (RREs) is critical to enhance our understanding of PTR. It offers new opportunities for understanding both gene regulation and consequences of genetic variation in transcript regions aside from the open reading frame.
We recently developed a protocol for the transcriptome-wide isolation of RREs readily applicable to any protein or RNP directly contacting RNA (including RNA helicases, polymerases, or nucleases) expressed in cell culture models either naturally or ectopically (Hafner, Landthaler et al. 2010).
Briefly, immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. In the course of lysate preparation and immunoprecipitation, the mRNAs are partially degraded using Ribonuclease T1. The isolated crosslinked RNA fragments are converted into a cDNA library and deep-sequenced using Solexa
technology. By introducing photoreactive nucleosides that generate characteristic sequence changes upon crosslinking (see below), our protocol allows one to separate RNA segments bound by the protein of interest from the background un-crosslinked RNAs.
Post-transcriptional regulation (PTR) of messenger RNAs (mRNAs) plays important roles in diverse cellular processes (Ambros 2004; Halbeisen, Galgano et al. 2008). The fates of mRNAs are determined predominantly by their interactions with RNA-binding proteins (RBPs) and non- coding, guide-RNA-containing ribonucleoprotein complexes (RNPs). Taken together, they form mRNA-containing ribonucleoprotein complexes (mRNPs). The RBPs influence the structure and interactions of the RNAs and play critical roles in their biogenesis, stability, function, transport and cellular localization (Moore 2005; Keene 2007; Glisovic, Bachorik et al. 2008).
Given that hundreds of RBPs and RNPs and their networks remain to be studied and evaluated in a cell-type-dependent manner, the development of powerful tools to determine their binding sites or RNA recognition elements (RREs) is critical to enhance our understanding of PTR. It offers new opportunities for understanding both gene regulation and consequences of genetic variation in transcript regions aside from the open reading frame.
We recently developed a protocol for the transcriptome-wide isolation of RREs readily applicable to any protein or RNP directly contacting RNA (including RNA helicases, polymerases, or nucleases) expressed in cell culture models either naturally or ectopically (Hafner, Landthaler et al. 2010).
Briefly, immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. In the course of lysate preparation and immunoprecipitation, the mRNAs are partially degraded using Ribonuclease T1. The isolated crosslinked RNA fragments are converted into a cDNA library and deep-sequenced using Solexa
technology. By introducing photoreactive nucleosides that generate characteristic sequence changes upon crosslinking (see below), our protocol allows one to separate RNA segments bound by the protein of interest from the background un-crosslinked RNAs.
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