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Rockefeller Team Maps MicroRNA Interactions in Mouse Brain

NEW YORK (GenomeWeb News) – A research team from Rockefeller University has developed a protocol for mapping microRNA-messenger RNA interactions — an approach that they validated in mouse brain tissues.

The researchers used high-throughput RNA sequencing of cross-linked immunoprecipitation, or HITS-CLIP, to evaluate RNA molecules interacting with the argonaute protein Ago. By pulling together Ago-miRNA and Ago-mRNA interaction maps using bioinformatics, they demonstrated that they could extrapolate mRNA-miRNA binding patterns in the mouse brain from the two data sets. The work appeared in the advanced, online edition of Nature this week.

"It is thought that RNA is the molecule that can explain the gap between the complexity of cellular functions and our limited number of genes," senior author Robert Darnell, a Howard Hughes investigator and molecular neuro-oncology researcher at Rockefeller University, said in a statement. "We now have a platform to evaluate the degree to which microRNAs contribute to this complexity with an extraordinary amount of precision."

MicroRNAs, short nucleotides that are around 20 bases long, help regulate gene expression through the RNA induced silencing complex, or RISC, pathway, targeting specific mRNAs over six to eight nucleotides.

But since potential miRNA binding sites are peppered throughout the genome, it's not always easy to tell where miRNAs bind or what mRNAs they target. And, the authors noted, algorithms used to find these binding sites are prone to false-positive results, making it difficult to predict miRNA-mRNA interactions.

To circumvent such challenges, Darnell and his team exploited the properties of a RISC component — the argonaute protein Ago, which interacts with both miRNAs and mRNAs. By cross-linking the protein to the RNA, doing immunoprecipitation experiments, and sequencing bound RNAs, they could then get a snapshot of where miRNAs and mRNAs overlapped.

Members of Darnell's team developed the cross-linking and immunoprecipitation method for investigating protein-RNA interactions several years ago. HITS-CLIP extends the approach, using high-throughput sequencing to identify RNAs of interest.

"HITS-CLIP offers a clear means of identifying direct Ago targets and identifying specific interaction sites, which in turn offers the possibility of specifically targeting miRNA activity," the authors wrote.

The researchers used ultraviolet-irradiation to cross-link protein and RNAs in mouse brain samples. They then did immunoprecipitation, labeled and purified Ago-RNA complexes, and used the Illumina Genome Analyzer to sequence the Ago-bound miRNA or mRNA.

The team repeated the protocol several times with different antibodies to decrease the risk of picking up non-specific interactions and found 454 different miRNAs in the mouse brain tissue.

By comparing different replicates, the team narrowed in on sites most likely to represent true interactions, identifying nearly 1,500 Ago-mRNA clusters on 829 transcripts in the mouse brain.

The pattern of these interactions suggested that nearly a third of Ago-mRNA interactions occur in 3' untranslated regions, though tags were also detected in coding sequences, introns, non-coding RNAs, and 5' UTR regions.

When they used linear regression analyses to look for Ago-mRNA CLIP sequences that might interact with miRNA sequences, the researchers found half a dozen motifs that were enriched in mRNA and also detected in the miRNAs binding Ago.

In particular, the researchers noted that the seed sequence for a brain specific miRNA called miR-124 seemed to be over-represented in the mouse genome. Their subsequent experiments suggested HITS-CLIP data for miR-124 was consistent with previous miR-124 functional studies, bolstering the team's confidence that the HITS-CLIP was picking up authentic interactions.

When they mapped binding patterns for the 20 most abundant Ago-miRNA clusters, the team found that these miRNAs tended target genes involved in neuronal differentiation and cytoskeleton regulation-related pathways, based on gene ontology analyses.

And, researchers say, the general approach used in the paper should be useful for mapping and characterizing miRNA-mRNA interactions in other systems as well.

"Here, we use Ago HITS-CLIP to define the sites of Ago interactions in vivo, decoding a precise map of miRNA-mRNA interactions in the mouse brain," Darnell and his co-authors wrote. "This provides a platform that can establish the direct targets on which miRNAs act in a variety of biological contexts, and the rules by which they do so."

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