Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP.

RNA transcripts are subject to posttranscriptional gene regulation involving hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) expressed in a cell-type dependent fashion. We developed a cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs. The crosslinked sites are revealed by thymidine to cytidine transitions in the cDNAs prepared from immunopurified RNPs of 4-thiouridine-treated cells. We determined the binding sites and regulatory consequences for several intensely studied RBPs and miRNPs, including PUM2, QKI, IGF2BP1-3, AGO/EIF2C1-4 and TNRC6A-C. Our study revealed that these factors bind thousands of sites containing defined sequence motifs and have distinct preferences for exonic versus intronic or coding versus untranslated transcript regions. The precise mapping of binding sites across the transcriptome will be critical to the interpretation of the rapidly emerging data on genetic variation between individuals and how these variations contribute to complex genetic diseases.

In vivo and transcriptome-wide identification of RNA binding protein target sites.

Animal mRNAs are regulated by hundreds of RNA binding proteins (RBPs). The identification of RBP targets is crucial for understanding their function. A recent method, PAR-CLIP, uses photoreactive nucleosides to crosslink RBPs to target RNAs in cells prior to immunoprecipitation. Here, we establish iPAR-CLIP (in vivo PAR-CLIP) to determine, at nucleotide resolution, transcriptome-wide binding sites of GLD-1, a conserved, germline-specific translational repressor in C. elegans. We identified 439 reproducible target mRNAs and demonstrate an excellent dynamic range of target detection by iPAR-CLIP. Upon GLD-1 knockdown, protein but not mRNA expression of the 439 targets was specifically upregulated, demonstrating functionality. Finally, we discovered strongly conserved GLD-1 binding sites near the start codon of target genes. These sites are functional in vitro and likely confer strong repression in vivo. We propose that GLD-1 interacts with the translation machinery near the start codon, a so-far-unknown mode of gene regulation in eukaryotes.

TatB functions as an oligomeric binding site for folded Tat precursor proteins.

Twin-arginine-containing signal sequences mediate the transmembrane transport of folded proteins. The cognate twin-arginine translocation (Tat) machinery of Escherichia coli consists of the membrane proteins TatA, TatB, and TatC. Whereas Tat signal peptides are recognized by TatB and TatC, little is known about molecular contacts of the mature, folded part of Tat precursor proteins. We have placed a photo-cross-linker into Tat substrates at sites predicted to be either surface-exposed or hidden in the core of the folded proteins. On targeting of these variants to the Tat machinery of membrane vesicles, all surface-exposed sites were found in close proximity to TatB. Correspondingly, incorporation of the cross-linker into TatB revealed multiple precursor-binding sites in the predicted transmembrane and amphipathic helices of TatB. Large adducts indicative of TatB oligomers contacting one precursor molecule were also obtained. Cross-linking of Tat substrates to TatB required an intact twin-arginine signal peptide and disappeared upon transmembrane translocation. Our collective data are consistent with TatB forming an oligomeric binding site that transiently accommodates folded Tat precursors.

PAR-CliP--a method to identify transcriptome-wide the binding sites of RNA binding proteins.

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.