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Delaware's Green Builds on RNA-seq for Tool to Study MicroRNA Targets

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A flurry of recent work has used RNA-seq to analyze  whole transcriptomes of model organisms and humans.

Using that technology as a foundation, a new technique, detailed in work recently published in Nature Biotechnology, has winnowed down identifiable transcripts to microRNA cleavage products to find both these targets and their corresponding miRNAs.

Using a combination of rapid amplification of cDNA ends and computational analysis to sift through the entirety of transcripts, Pam Green at the Delaware Biotechnology Institute at the University of Delaware looked across a specific subset of miRNA cleavage products to identify, clone, and then match target messages back to reference small RNAs. "This is looking at a particular kind of end," Green says. "RNA-seq is looking more to quantitate the RNA." She's called her technique PARE, or parallel analysis of RNA ends.

In plants, one way an miRNA can cleave its target mRNA is by generating a truncated RNA product with a 5' phosphate end. Green used this signal to identify, clone, and amplify only these mRNAs. Uncleaved RNA would have a "cap," she says, "so it wouldn't clone with our procedure. We were then able to use all that data to find which RNAs had the kind of cleavage event that would match [back] to a small RNA."

One of her tricks was using an Arabidopsis mutant that lacked XRN4 activity. XRN4 is an enzyme that typically degrades uncapped mRNAs; the mutant plant, however, couldn't send these molecules packing, so cleavage products accumulated at a much higher level than in normal plants. Effectively, this increased the sensitivity of detection. "The sensitivity jumped tremendously — it would amplify the accumulation of all those cleavage products," Green says.

The team's process not only allowed researchers to experimentally identify new targets and validate many predicted ones, but also to find new miRNAs. "By matching them back to the small RNA, it had an additional application that goes way beyond what RNA-seq would do," Green says. "It allowed us to find new microRNAs by essentially doing a reverse of what you would call a target prediction. You could reverse that process and get the microRNA that could generate that target cleavage."

Additionally, she says, "this kind of approach could be used in animal cells to find other cases of microRNA-directed cleavage that might be rare but extremely important." The approach doesn't have to be limited to look at only RNAs with 5' phosphate ends. Green says her method could be used to look at other kinds of ends in order to study "other aspects of post-transcriptional control."

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