The protein translation is a complicated process. In the past decades, scientists have focused mainly on the interpretation of the coding region of a gene, which can directly determine the amino acid composition of a protein. Recently, research on the non-coding RNA (ncRNA) regions have greatly interested the scientists and large amount of evidence indicates that ncRNAs vibrantly participate in the translational process, especially non-sequence-dependent epigenetic modifications, RNA-RNA bind protein (RBP) interactions, and post-transcriptional regulation.
Many long non-coding RNAs (lncRNAs) have been identified while the functions of which are still not clear. To further investigate RNA-RBP interaction as a critical mechanism regulating the translation, scientists have developed various technologies to address these questions. The most representative technologies are RNA immunoprecipitation sequencing (RIP-Seq) and crosslinking-immunoprecipitation sequencing (CLIP-Seq), both of which use antibodies of RBPs to pull down binding RNAs but vary slightly in principles and detailed protocols.
RIP-seq
RIP-Seq combines RNA immunoprecipitation and high-throughput sequencing, in which the interacting RNA is captured through immunoprecipitation of target proteins. High-throughput sequencing of the captured RNA helps to understand the dynamic process of the post-transcriptional regulatory network.
Advantages
1. RIP-Seq is versatile in interpreting RNA-RBP interaction network with regard to various ncRNAs, such as miRNA and lncRNA.
2. Compared with CLIP-Seq, RIP-SEQ does not require conducting UV cross-linking, which makes it easy to operate and guarantee repeatable and accurate results.
3. With wide coverage, protein-binding sites can be screened and identified at the genome-wide range.
4. With high resolution and high-throughput sequencing technology, new protein binding sites can be found.
Applications
1. Verification of the interaction between RNA and target protein
2. Genome-wide identification of RNA-RBP interaction networks
3. Analysis of the interaction between RBP and miRNA, lncRNA, and other ncRNAs
CLIP-Seq
The UV cross-linking and immunoprecipitation (CLIP) is another widely used method for the in vivo study of binding pattern between RBPs and numerous RNA targets, which reveals the biological functions of the binding process. The main principle is based on the covalent binding between RNA molecules and RBPs under ultraviolet irradiation, which improves the binding strength of RNA binding proteins and corresponding RNA targets.
Advantages
1. High accuracy, faithfully reflecting the interaction among molecules in vivo.
2. Strong specificity: the ultraviolet radiation does not cause protein-protein cross-linking while being able to identify the interaction between RBPs and RNA directly.
3. Wide application range: especially suitable for research on splicing factors, RNA binding proteins, miRNA targets as well as their interactions.
Applications
1. Verification of the interaction between RNA and target protein directly as well as the exact binding sites
2. Genome-wide identification of RNA-RBP interaction networks
3. Identification and functional mechanism research of lncRNA, circRNA, miRNA, etc.
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