Together, these problems introduce significant artifacts that make downstream analysis challenging. This bias leads to overrepresentation of sequences not bound by the RNA-binding protein in the final library. This frequent early termination on crosslinked templates results in a bias towards RNAs that do not have crosslinking sites. These crosslinked bases are frequent sites of termination during reverse transcription, meaning that only a fraction of reverse transcription events on crosslinked RNAs will read through to the 5′ adaptor and produce PCR-competent templates. Recovery of irrelevant misprimed sequences is exacerbated by the fact that proteinase K digestion leaves one or two amino acids crosslinked to the RNA. These cDNAs, which will be included in final sequencing libraries if they received a 5′ adaptor, contaminate the library with read pileups anchored to genomic occurrences of the adaptor sequence, rather than bona fide sites of protein binding. Such mispriming events produce cDNAs from RNAs that may lack 3′ adaptor sequences. The use of a perfectly complementary reverse transcription primer results in substantial mispriming to suboptimal matches of the 5′ end of 3′ adaptor sequence in alternative RNAs during reverse transcription. A 5′ RNA linker is then ligated to the purified RNAs, and the RNA is reverse-transcribed using a primer perfectly complementary to the 3′ adaptor. The resulting size-selected protein-RNA complexes are then digested with Proteinase K to remove the protein and the remaining RNA is purified by phenol extraction and ethanol precipitation. The RNAs are then treated with PNK, again “on-bead”, and the protein-RNA complexes are resolved on an SDS-page gel to select only complexes of the expected size. This step is performed “on-bead”, and the efficiency is highly dependent on the structure of the RNA-adaptor interaction. The protein-bound RNAs are then dephosphorylated and a 3′ RNA adaptor is ligated with T4 RNA ligase. HITS-CLIP library construction begins with the cross-linking of RNA-binding proteins to RNA using UV light, partial RNA digestion with RNAse A, and immunoprecipitation of the RNA-binding protein of interest and attached RNAs. However, while useful, these techniques suffer from substantial mispriming during reverse transcription that can contaminate the resulting libraries with false peaks that may be difficult to distinguish from physiologically relevant peaks. These techniques have made important contributions to the understanding of the function of a range of RNA-binding proteins, from the microRNA-related argonaute (AGO) to other regulatory factors, including FUS and PTBPs.
The advent of HITS-CLIP and its derivative techniques has facilitated the mapping of RNA-protein interactions genome-wide in a variety of tissues and organisms. Here, we present a modification to the HITS-CLIP protocol that effectively eliminates this artifact and improves the sensitivity and complexity of resulting libraries. We also found that standard techniques for validating microRNA-target interactions fail to differentiate between artifactual peaks and physiologically relevant peaks. Up to 45 % of peaks in publicly available HITS-CLIP libraries are attributable to this mispriming artifact, and the majority of libraries have detectable levels of mispriming. We found that substantial mispriming during reverse transcription results in the overrepresentation of sequences complementary to the primer used for reverse transcription. Hence, the accuracy and sensitivity of binding site identification is critical. High-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP) allows for high resolution, genome-wide mapping of RNA-binding proteins. This methodology is frequently used to validate predicted targets of microRNA binding, as well as direct targets of other RNA-binding proteins.
0 kommentar(er)
