MicroRNAs and other small RNAs enriched in the Arabidopsis RNA-dependent RNA polymerase-2 mutant

Abstract

The Arabidopsis genome contains a highly complex and abundant population of small RNAs, and many of the endogenous siRNAs are dependent on RNA-Dependent RNA Polymerase 2 (RDR2) for their biogenesis. By analyzing an rdr2 loss-of-function mutant using two different parallel sequencing technologies, MPSS and 454, we characterized the complement of miRNAs expressed in Arabidopsis inflorescence to considerable depth. Nearly all known miRNAs were enriched in this mutant and we identified 13 new miRNAs, all of which were relatively low abundance and constitute new families. Trans-acting siRNAs (ta-siRNAs) were even more highly enriched. Computational and gel blot analyses suggested that the minimal number of miRNAs in Arabidopsis is approximately 155. The size profile of small RNAs in rdr2 reflected enrichment of 21-nt miRNAs and other classes of siRNAs like ta-siRNAs, and a significant reduction in 24-nt heterochromatic siRNAs. Other classes of small RNAs were found to be RDR2-independent, particularly those derived from long inverted repeats and a subset of tandem repeats. The small RNA populations in other Arabidopsis small RNA biogenesis mutants were also examined; a dcl2/3/4 triple mutant showed a similar pattern to rdr2, whereas dcl1-7 and rdr6 showed reductions in miRNAs and ta-siRNAs consistent with their activities in the biogenesis of these types of small RNAs. Deep sequencing of mutants provides a genetic approach for the dissection and characterization of diverse small RNA populations and the identification of low abundance miRNAs.

Figure 1.

Small RNAs or clusters common to wild type and rdr2. Venn diagrams representing genome-matched rdr2 454 and MPSS sequences from Table 1. (A) A comparison of distinct signatures in the MPSS libraries indicates 19% of rdr2 sequences were also found in wild type. (B) A comparison of distinct signatures in the 454 libraries indicates 21% of rdr2 sequences were also found in wild type. (C) A comparison of genomic clusters of MPSS signatures indicates 93% of small RNA clusters represented in rdr2 were also found in wild type. For this analysis, clusters contained at least three small RNAs across both libraries; this cutoff was chosen arbitrarily to remove clusters with only one or two small RNAs that could be background. Most of the rdr2-only clusters are low-abundance miRNAs or other “real” sequences that were not detected due to depth of coverage in the wild-type library.

Figure 2.

Chromosomal distribution of small RNAs from wild-type and rdr2 inflorescence. Inflorescence small RNAs matched to the Arabidopsis chromosomes as measured by MPSS. The height of the vertical lines indicates the abundance of the small RNA. Maximum height of black bar, >25. The red bars indicate signatures with >125 transcripts per quarter million (TPQ) and highest black bars indicate signatures with >25 and ≤125 TPQ; other black bars indicate signature with ≤25 TPQ. Horizontal green bars indicate the approximate location of the centromere. (A) Wild-type inflorescence (adapted from Lu et al. 2005). (B) rdr2 inflorescence; the abundance of known miRNAs is indicated by blue lines.

Figure 3.

Use of rdr2 sequences to select miRNA candidates from previously identified wild-type small RNAs. Five-way Venn diagram of selection criteria for miRNAs. The number of distinct rdr2 MPSS signatures matching the criteria is indicated in each box numbered in upper right; only rdr2 signatures also found in the wild-type library are represented. The figure excludes 13,153 distinct signatures that did not pass any of the criteria (of which 1583 were found in both rdr2 and wild-type inflorescence libraries) and 54 matching to the criteria in the Venn which were present in rdr2 but not wild-type inflorescence (these 54 are included in Supplemental Fig. S2). The paired, sparse, abundance filters, and AtSet1 and AtSet2 filters are described elsewhere (Jones-Rhoades and Bartel 2004; Lu et al. 2005) but represent potential hairpin structures typical of miRNA precursors and conservation of those structures in rice, respectively.

Figure 4.

Novel miRNAs identified from Venn analysis of rdr2 sequences. Small RNAs were selected for validation by RNA gel blots, as described in the text. Low molecular weight RNA isolated from inflorescence tissues was probed with labeled oligonucleotides. The lanes in the blots include the following samples: wild type, rdr2, rdr6, dcl1–7, and dcl2/3/4. The normalized abundance level from the MPSS data for rdr2 and wild type is listed to the right of the identifier for each small RNA. The reason for the apparent increases in abundance in rdr2 vs. wild type in the blots is not clear; approximately equal amounts of RNA were loaded. It does not appear to be due to RDR2-dependent small RNAs in the 5′ flanking regions, although for miR775, the most extreme case, there is an overlapping small RNA that is largely RDR2-dependent that might interfere with miR775 production in wild type.

Figure 5.

Estimating the minimal Arabidopsis miRNA population. (A) A Venn diagram to identify the number of miRNAs that may exist in Arabidopsis. Numbers in each section indicate sequences that satisfy each criterion, include AtSet1 hairpin folds (Jones-Rhoades and Bartel 2004), known miRNAs (underlined numbers), and genomic clusters of rdr2 MPSS signatures (numbers in brackets). AtSet1 and rdr2 clusters were considered matching if the start or end of either overlapped. Lowercase Roman numerals indicate sections from which sequences were selected in B. (B) Gel blots to evaluate the fraction of bona fide miRNAs in selected sectors. Small RNAs identified by rdr2 signatures in section (i) in A were randomly selected and evaluated by RNA gel blots to determine their presence/absence in the dcl1–7, dcl2/3/4, and rdr6 mutants. LNA probes were used for miR782, miR783, small86, and small88 (see Methods).

Figure 6.

RDR2-independent small RNAs from regions with ta-siRNA-like features. (A) The locus that includes small49 exhibits 21-nt phasing and accumulation characteristics in mutants similar to those of ta-siRNAs. Image of the MPSS Web viewer for the intergenic region that contains small49 in the position indicated by the large gray arrow (the shaded square covering both strands in this intergenic region indicates an inverted repeat); an RNA gel blot of small49; a plot of the y-axis indicating the small RNA abundance in the rdr2 mutant as measured by MPSS (in TPQ), and the x-axis indicating nucleotide position on Chr. 1, with the “697” indicating position 25,282,697. (B) The blot shows that small58 also has ta-siRNA-like accumulation features; images as described in A, with the 0 position in the x-axis of the plot indicating nucleotide 13,295,900 on Chr. 4. Due to limited space, only a portion of this intergenic region is shown, with the flanking genes indicated at the ends by their At identifiers.

Figure 7.

Small RNA size distribution in mutants evaluated with 454 sequencing. In each plot, gray indicates wild type, light blue is rdr2, green is dcl1–7, dark blue is rdr6, and red is the dcl2/3/4 triple mutant. (A) Number of distinct signatures vs. size. (B) Total abundance of sequences vs. size. (C) Number of distinct vs. size, with known miRNAs removed. (D) Total abundance vs. size, with known miRNAs removed.