Protein binding is involved in every step of the transcript life cycle, from synthesis (polymerases) to degradation (nucleases). A subset of these transcripts is involved in other critical processes such as epigenetic regulation and genome protection through transposon silencing, in addition to protein synthesis. The majority of research has focused on transcriptomics profiling. However, it is thought that mRNA levels do not always directly correlate with steady-state protein levels. As the importance of RNA processing and translational events that occur post-transcriptionally becomes clearer, researchers are increasingly interested in identifying the RNAs associated with RNA binding protein (RBP) in a cellular context.
Within RNA-protein complexes, RIP-Seq maps the sites where proteins are bound to the RNA. The purification of RNA–protein interactions in native conditions using a protein-specific antibody to map the RBP of interest is referred to as RNA immunoprecipitation (RIP). With the advent of sequencing technologies and various RIP chemistries, thousands of bound transcripts (mRNAs, non-coding RNAs, or viral RNAs) can now be detected simultaneously in a single experiment.
(1) RIP-Seq is versatile in analyzing RNA-RBP interaction networks with regard to various ncRNAs, such as miRNA and lncRNA, (2) RIP-Seq does not necessitate undertaking UV cross-linking, which makes it simple to function and ensure repeatable and accurate results, (3) RIP-Seq does not necessitate conducting UV cross-linking, which makes it easy to perform and assure repeatable and accurate results, RIP-Seq does not require conducting UV (3) Protein-binding sites can be screened and recognized across the genome with wide coverage, and (4) new protein binding sites can be discovered with high resolution and high-throughput sequencing technology.
However, it has the following drawbacks: (1) it needs antibodies to the targeted proteins, (2) nonspecific antibodies will precipitate nonspecific complexes, (3) lack of crosslinking or stabilization of the complexes may result in false negatives, and (4) RNase digestion must be carefully monitored.
Figure 1. RNA immunoprecipitation sequencing protocol workflow. (Gagliardi, 2016)
Before preparing genomic DNA fragments for sequencing, RIP-Seq uses antibody capture to enrich relevant sequences. Within RNA-protein complexes, RIP-Seq maps the sites where proteins are bound to the RNA. RNA-protein complexes are immunoprecipitated with antibodies specific for the protein of interest in this method. Following RNase digestion, protein-bound RNA is extracted and reverse-transcribed to cDNA. After that, the locations can be traced back to the genome. The single-base resolution of protein-bound RNA is achieved through deep sequencing of cDNA. The RIP-Sequencing protocol is summarized as follows:
1. Collect cells (optional treatment of cells with formaldehyde to cross-link in vivo protein-RNA complexes)
2. Isolate nuclei from nuclear pellets and lyse them
3. chromatin shear
4. Immunoprecipitate the target RNA binding protein (RBP) along with the bound RNA.
5. Purify RNA bound to immunoprecipitated RBP after washing off unbound material.
6. Convert RNA to cDNA and analyze using qPCR, microarrays, or sequencing.
The interacting RNA is captured through immunoprecipitation of target proteins in RIP-Seq, which combines RNA immunoprecipitation and high-throughput sequencing. The dynamic process of the post-transcriptional regulatory network can be better understood with high-throughput sequencing of the captured RNA. The following are some of its applications: (1) verification of RNA-target protein interactions, (2) genome-wide identification of RNA-RBP interaction networks, and (3) analysis of RBP interactions with miRNA, lncRNA, and other ncRNAs.