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RIP-Chip Tears Into miRNP Complex Analysis

January 6, 2010 Solving the mysteries of miRNA:mRNA interactions has been a tough case to crack. Correlation studies, and miRNA target prediction algorithms point us in the right direction, but their indirect nature is a little like going to trial with only circumstantial evidence. Researchers from University of Kentucky published a new paper demonstrating how RIP-Chip techniques can provide direct, CSI-worthy evidence to help investigate how microribonucleoprotein (miRNP) complexes really work.

The team from Kentucky started off their experiments by using an anti-argonaute (AGO) antibody to pull down miRNPs from the H4 cell-line they were studying, and from H4 cells transfected with miR-107, miR-124, miR-128, miR-320 or a control miRNA; and then running the IP’d RNA on a microarray to look for miRNA targets. Here’s what they learned:

• Rip-Chip can accurately find miRNA:mRNA interactions
• RIP-Chip can identify primary (miRNP related) versus secondary or “downstream” miRNA regulation
• miRNA transfections sometimes alter the mRNAs that are incorporated into miRNPs
• Those specific mRNAs found in miRNPs after transfection only moderately agree with computational target predictions
• Some miRNAs (miR-107 and miR-128) caused exclusion of mRNAs normally associated with miRNPs
• miR107 and miR-128 transfections caused lower AGO levels, but AGO was not found in miRNPs, indicating that a secondary miRNA regulation can occur without a stable mRNA:miRNP association.

The authors conclude that RIP-Chip is a powerful, direct and high throughput tool to look at miRNA:mRNA relationships and global miRNA regulation.

Go over all the evidence in RNA, January 2010