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Abstract
MicroRNAs (miRNAs) regulate the expression of target mRNAs in plants and animals [1].
Plant miRNA targets have been predicted on the basis of their extensive and often
conserved complementarity to the miRNAs [2-4], as well as on miRNA overexpression
experiments [5]; many of these target predictions have been confirmed by isolation
of the products of miRNA-directed cleavage. Here, we present a transcriptome-wide
experimental method, called "degradome sequencing," to directly detect cleaved miRNA
targets without relying on predictions or overexpression. The 5' ends of polyadenylated,
uncapped mRNAs from Arabidopsis were directly sampled, resulting in an empirical snapshot
of the degradome. miRNA-mediated-cleavage products were easily discerned from an extensive
background of degraded mRNAs, which collectively covered the majority of the annotated
transcriptome. Many previously known Arabidopsis miRNA targets were confirmed, and
several novel targets were also discovered. Quantification of cleavage fragments revealed
that those derived from TAS transcripts, which are unusual in their production of
abundant secondary small interfering RNAs (siRNAs), accumulated to very high levels.
A subset of secondary siRNAs are also known to direct cleavage of targets in trans[6];
degradome sequencing revealed many cleaved targets of these trans-acting siRNAs (ta-siRNAs).
This empirical method is broadly applicable to the discovery and quantification of
cleaved targets of small RNAs without a priori predictions.