Endogenous siRNA and miRNA Targets Identified by Sequencing of the Arabidopsis Degradome
Charles Addo-Quaye,1 Tifani W. Eshoo,2 David P. Bartel,4,5
and Michael J. Axtell2,3,*
1Department of Computer Science and Engineering
2Cell and Developmental Biology Graduate Program
Huck Institutes of the Life Sciences
3Department of Biology
Pennsylvania State University
University Park, Pennsylvania 16802
4Whitehead Institute
Cambridge, Massachusetts 02142
5Howard Hughes Medical Institute and Department of Biology
Massachusetts Institute of Technology
Cambridge, Massachusetts 02139
Summary 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 50 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.
