Endogenous siRNA and miRNA Targets Identified by Sequencing of the Arabidopsis Degradome

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.

MicroRNAs in tomato plants

“…Several miRNAs have multitarget genes involved in a range of transcription factors, metabolic processes, stress defense and signaling. Validation of miRNA targets by 5′-RACE is achieved by miRNA-guided cleavage at the transcriptome level [75]. Plant miRNAs regulate numerous target genes and several miRNAs share the downstream targets.…”

The use of high-throughput sequencing methods for plant microRNA research

“…55,56 All of these methods take advantage of the fact that T4 RNA ligase 1 is utilized for sequencing library construction by ligating an adapter to the 5 0 end of the poly(A)-tailed transcript with a free 5 0 -monophosphate. Thus, the intact mature mRNAs with 5 0 caps will not be included for library preparation.…”

microRNAs in a multicellular green alga Volvox carteri

“…Degradome libraries were constructed as described [16,17]. After discarding low-quality reads and removing adaptor sequences, signatures were mapped to genome sequences and mapped reads were treated as dataset for Cleaveland [18].…”

“…Next, we employed a high-throughput "degradome" sequencing approach [16,17] to validate the predicted miRNA targets. We constructed the cDNA library for degradome sequencing as described [16].…”

“…We constructed the cDNA library for degradome sequencing as described [16]. High-throughput sequencing resulted in 6005274 reads that match to the transcriptome of Volvox (http://genome.jgi-psf.org/Volca1/Volca1.download.…”