In fact, the microRNAs direct enzymes to cut the messenger RNAs in two. The resulting decay products--or "cell trash," as German refers to them--are the focus of the UD technique.
Rather than searching for the cleaved RNAs one by one in a tedious process that previously had been the standard, the UD technique collects all of this "cell trash" to analyze at once, saving researchers time and labor, while generating a rich data set for analysis.
"We tried to find all of the microRNA-target RNA pairs at once," German said. "MicroRNAs have a broad impact on gene regulation, but what those regulated genes are was the first focus."
The approach captures the ends of the decaying messenger RNA and the location at which the molecule has been cut. Because the cleaved molecules are recognizable by the high level of the specific cut site in the libraries generated by the UD technique, they can be distinguished from molecules decayed by other cellular processes. Meyers' lab, led by Manoj Pillay, a master's student from the Department of Computer and Information Sciences, developed bioinformatics techniques for sorting through the different signals.
Illumina Inc., in Hayward, Calif., analyzed some 28 million sequences for the study, using a high-throughput technique known as sequencing by synthesis (SBS). The application of this technique to the UD-made libraries produced a distinct "signature" for each cleaved messenger RNA molecule, which can be assigned to its respective target gene.
In an interesting twist, the researchers discovered messenger RNA targets for which no microRNAs were known to match or cause decay. This led them back to their database of small RNAs, from which they identified four new microRNAs in Arabidopsis, boosting the total to 183.
"This research validated both known and new messenger RNA targets of microRNAs," Meyers noted. "The approach will enable us to analyze RNA degradation products at a very l
|Contact: Tracey Bryant|
University of Delaware