New technology to isolate and analyze intact RBP-mRNA-miRNA complexes.

The new miRNA RIP-chip system is designed to identify miRNAs bound to their mRNA targets, analyze the maturation mechanism of miRNAs, identify RISC components and analyze their functions.

Scientific Background

Discovery of RNA interference (RNAi) has given a great boost to research on functional RNA and post-transcriptional regulation. RNAi, which plays a central role in sequence-specific gene silencing in eukaryotic cells, depends on the functions of RNA-induced silencing complex (RISC) composed of small non-coding RNAs (ncRNAs) and proteins. The main classes of small ncRNA are short interfering RNAs (siRNAs), microRNAs (miRNAs) and PIWI-interacting RNAs (piRNAs). siRNAs are generated by cleavage of exogenous long double-stranded RNA precursors in response to viral infection or artificial introduction. In contrast, miRNAs are generated from endogenous transcripts containing stem-loop structures. The siRNAs and miRNAs processed by Dicer, which functions as a ribonuclease III enzyme, are incorporated into the RISC in order to silence the specific mRNAs based on partial sequence complementarity between the small RNAs and the 3’ untranslated regions (UTRs) of the mRNAs. Argonaute family proteins are a core component of RISC and divided into the AGO and PIWI subfamilies. siRNAs and miRNAs are loaded onto AGO proteins whereas piRNAs are loaded onto PIWI proteins. Each member of the family proteins functions as a silencer to inactivate their target mRNAs.

Hundreds of miRNA species have been discovered in animals and plants, many of which exhibit temporally and spatially controlled expression. One approach to investigate the biological functions of miRNAs has been to identify their targets. The miRNA target predictions are based on computational analyses of complementary sequence elements that are refined by considering evolutionary homologies across multiple species. While a variety of computational approaches and algorithms have been used to make such predictions, it is not certain that each miRNA gains functional access to these targeted mRNAs in the cell under a given set of conditions.

RIP-Chip (ribonucleoprotein immunoprecipitation-microarray profiling) is a biochemical approach to identify the composition and organization of endogenous mRNAs, miRNAs and RNA binding proteins (RBPs) within messenger ribonucleoprotein (mRNP) complexes. RIP-Chip has been successfully employed to isolate AGO-containing RNPs by immunopurification with anti-AGO antibodies. When the co-isolated miRNA and mRNA subpopulations are analyzed using the computational predictions of conserved seed sequences, this approach provides a powerful tool to identify functional miRNA targets based on their physical interaction in vivo. RBPs have been reported to bind to mRNAs that encode functionally related proteins, and coordinately regulate these mRNAs during cellular processes. The RIP-Chip approach can isolate functionally related mRNAs. Since miRNAs can be co-immunoprecipitated with those mRNAs, the RIP-Chip approach can also isolate miRNAs that regulate specific group of mRNAs that are functionally related.

RIP Assay Kits

RIP Validated Antibodies

Antibodies Validated only for WB/IPP