RNA binding protein (RBP) plays an important role in the coordination of different levels of gene expression control and post-transcriptional regulation. Next generation sequencing (NGS) technology has opened up an unprecedented new way for the study of molecular biology and genetics. In addition to straightforward application in de novo sequencing or resequencing of genomes, genomic loci or transcripts, they allow the study of RBPs extracted by RNA immunoprecipitation (RIP) to be given RBP sequence analysis (RIP-Seq, RNA-immunoprecipitation followed by high-throughput sequencing) and characterization of all RNAs combined, with excellent results.
Ribonucleoprotein (RNP) complexes are formed by the binding of RNA and RBPs in cells. RBP mediates key RNA based processes, such as alternative splicing, polyadenylation, subcellular localization, translation, and miRNA regulation. Mutations in these proteins can lead to a variety of human diseases and disorders. Techniques for testing RNA protein interaction have been developed from DNA protein interaction techniques with the same principles, and RIP is one of them.
Eukaryotic genomes can encode hundreds of RBPs (thousands in vertebrates), each of which has unique RNA binding specificity. Since RBP binding of the same RNA molecule has occurred thousands of times in the cell at the same time, once separated from the protein, the binding RNA fragment should be found in many copies, so as to be enriched in the extracted samples. The advanced sequencing technology innovates the transcriptome analysis of prokaryotes and eukaryotes. RNA sequence analysis (RNA-seq) is a large-scale parallel sequencing method based on cDNA, which has been widely used in transcription boundary annotation. Therefore, RIP combined with NGS technology provides the most direct solution for the identification of DNA / RNA fragments enriched in samples (i.e., sequencing the samples themselves).
Fig.1 RIP-seq determines bound RNAs by analyzing immunoprecipitated RNPs by high-throughput sequencing. POI, protein of interest. (Moore, 2019)
Generally speaking, the main problem of applying NGS technology to RIP samples is that at present, they are not allowed to sequence the whole RNA molecules obtained from IP, but only allowed to sequence the short segments at one or two 5' ends. The single sequencing channel of Illumina/Solexa platform has generated millions of sequence reads with a length of no more than 75-100 bps, which has become the preferred method for such experiments. Therefore, we have assembled the sequencing platform of Illumina/Solexa and the scientists in the field of bioinformatics and RNA. We can read useful information (i.e., the original RNA sequence combined by protein) from the short sequence, and obtain the effective and high-quality information of RNA protein interaction.
What's more attractive is that, according to the needs of customers, Creative Biolabs can also provide personalized testing services on RNA-protein interactions, to provide further strong support for the study of complex regulatory mechanisms of gene expression. For specific analysis, please do not hesitate to contact us for more details.
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