Endogenous siRNA and miRNA targets identified by sequencing of the Arabidopsis degradome.
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.
Addo-Quaye
Charles
C
Department of Computer Science and Engineering, Pennsylvania State University, University Park, Pennsylvania 16802.
Eshoo
Tifani W
TW
Cell and Developmental Biology Graduate Program, Huck Institutes of the Life Sciences, Pennsylvania State University, University Park, Pennsylvania 16802.
Bartel
David P
DP
Whitehead Institute, Cambridge, Massachusetts 02142; Howard Hughes Medical Institute and Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139.
Axtell
Michael J
MJ
Cell and Developmental Biology Graduate Program, Huck Institutes of the Life Sciences, Pennsylvania State University, University Park, Pennsylvania 16802; Department of Biology, Pennsylvania State University, University Park, Pennsylvania 16802. Electronic address: mja18@psu.edu.
D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism*; D035683:MicroRNAs / Q000378:metabolism*; D005810:Multigene Family; D012333:RNA, Messenger / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000378:metabolism*; D017423:Sequence Analysis, RNA
D035683:MicroRNAs; D012333:RNA, Messenger; D034741:RNA, Small Interfering