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.

Figure 1. Degradome tags were 3' biased and correlated with transcript abundance

(A) Histogram displaying the 5' positions of degradome tags from the indicated libraries relative to normalized transcript position. Tags were counted in one percent bins. For clarity, one tag that was extremely abundant in libraries three and four was omitted. (B-D) Relationships between microarray-derived estimates of transcript abundance (y-axis) and degradome tag abundance (x-axis) for the indicated libraries. RMA-normalized array values were from [12] and were matched according to the tissue source (Inflorescence, ATGE_29, B and C; Seedlings, ATGE_96, D). Transcripts with degradome tag abundances equal to or less than one were omitted. Lines represent best-fit linear regressions; r² values represent correlation coefficients.

Figure 2. Experimental identification of cleaved miRNA targets without predictions

(A) Schematic of methodology to find evidence of miRNA-mediated cleavage from degradome tags. (B) Density of 5' position of degradome tags corresponding to ARF17, a category I target. Tags aligned with the ninth through eleventh nucleotides of a miR160 complementary site were combined and shown in red. (C) As in B for SPL6, a category II target of miR156. (D) As in B for MYB13, a category III target of miR858. (E) Histogram displaying mean ratio of targets found using 30 cohorts of randomly permuted miRNAs to the number found using the annotated mature miRNA query data set at different alignment scores. Error bars represent one standard deviation. Alignment score thresholds are indicated for category I, II, and III targets. (F) Summary of 121 cleaved miRNA and/or ta-siRNA target sites found using degradome analyses. Details in Supplementary Table 2.

Figure 3

High accumulation levels of TAS cleavage products. Histogram displays abundance of degradome tags corresponding to miRNA-mediated cleavage sites; red indicates TAS genes, as labeled.

Figure 4. Independent confirmation of cleavage targets identified in the degradome

(A) Accumulation of confirmed miRNA and ta-siRNA targets, separated by category or by previous experimental knowledge, in various miRNA (dcl1−7, hen1−1, hst-15, hyl1−2) and siRNA (dcl2−1, dcl3−1, rdr1−1, rdr2−1, rdr6−15) mutants (microarray data from [2]). (B) Top: Density of 5' position of degradome tags corresponding to the NAC2 transcript. Tags aligned with the ninth through eleventh nucleotides of a miR164 complementary site were combined and shown in red. Bottom: Density of clones obtained via gene-specific 5'-RACE. Blue arrow indicates position of gene-specific oligo sequences. (C) As in B for At1g10120 and miR396.