A diverse and evolutionarily fluid set of microRNAs in Arabidopsis thaliana

Abstract

To better understand the diversity of small silencing RNAs expressed in plants, we employed high-throughput pyrosequencing to obtain 887,000 reads corresponding to Arabidopsis thaliana small RNAs. They represented 340,000 unique sequences, a substantially greater diversity than previously obtained in any species. Most of the small RNAs had the properties of heterochromatic small interfering RNAs (siRNAs) associated with DNA silencing in that they were preferentially 24 nucleotides long and mapped to intergenic regions. Their density was greatest in the proximal and distal pericentromeric regions, with only a slightly preferential propensity to match repetitive elements. Also present were 38 newly identified microRNAs (miRNAs) and dozens of other plausible candidates. One miRNA mapped within an intron of DICER-LIKE 1 (DCL1), suggesting a second homeostatic autoregulatory mechanism for DCL1 expression; another defined the phase for siRNAs deriving from a newly identified trans-acting siRNA gene (TAS4); and two depended on DCL4 rather than DCL1 for their accumulation, indicating a second pathway for miRNA biogenesis in plants. More generally, our results revealed the existence of a layer of miRNA-based control beyond that found previously that is evolutionarily much more fluid, employing many newly emergent and diverse miRNAs, each expressed in specialized tissues or at low levels under standard growth conditions.

Figure 1.

Newly identified Arabidopsis miRNAs. (A) Predicted secondary structures of miRNA hairpins highlighting the miRNA (red) and miRNA* species (blue). (B) The miRNA hairpins of A, shown in bracket notation with a tally of reads mapping to the hairpin and nucleotides colored as in A. (C) RNA blots demonstrating that accumulation of six detectable miRNAs depended on DCL1, not DCL2, DCL3, DCL4, RDR2, or RDR6. As a loading control, blots were stripped and reprobed for U6. (D) Sequencing frequencies of Arabidopsis miRNA families. Shown are cumulative plots for all Arabidopsis miRNA families (red squares), conserved families (violet diamonds), and all families plus the 40 sequenced candidates (gray triangles). Fourteen families, 11 of which were conserved, were sequenced at a frequency of greater than one per 1000 (dashed line).

Figure 2.

DCL4-dependent miRNAs in Arabidopsis. (A) RNA blots demonstrating that the accumulation of two detectable miRNAs depended on DCL4 and not on DCL1, DCL2, DCL3, RDR2, or RDR6. As a loading control, blots were stripped and reprobed for U6. Similar results were obtained with dcl4-1 and dcl4-2 alleles (data not shown). (B) Predicted secondary structures of the miRNA hairpins, with lines denoting the sequences mapping to the miRNA (top) and miRNA* (bottom) arm of each hairpin. The thickness and color of the lines correspond to the number of total reads representing each small RNA species, as indicated in the key. The two reads corresponding to the antisense of miR822 and the single read mapping to the antisense of miR839 are not depicted.

Figure 3.

Extended homology between miRNA genes and their predicted target genes, suggestive of common origin. (A) MIR841, which illustrates a pattern of extended homology (orange shading) resembling that observed previously for MIR161 and MIR163 and their respective targets (Allen et al. 2005). Segments corresponding to the mature miRNA (red) and miRNA* (blue) are indicated. The diagram (top) depicts the target gene in right–left polarity, and the alignment (bottom) depicts the target gene and miRNA* segment as their reverse complements (rc). Numbers indicate positions in the protein-coding gene (At2g38810), counting from its first annotated nucleotide. Nucleotides are shaded to indicate those shared by all (orange) or most (yellow) aligned sequences. (B) MIR826, for which extended homology suggested an evolutionary pathway whereby a later duplication creating the miRNA* arm was nested within an earlier duplication. The genomic proximity of the miRNA and target gene is shown in the top diagram. For the middle and bottom diagrams, drawing conventions are as in A. (C) MIR846, for which extended homology suggested tandem duplication within an ancestral gene whereby the duplicated regions independently gave rise to the miRNA and miRNA* segments.

Figure 4.

An intronic hairpin positioned so as to mediate DCL1 autoregulation. (A) Intron 14 of the DCL1 primary transcript, and the predicted hairpin structure of miR838. Arrows indicate 5′ ends of RACE-mapped fragments. (B) Alternative fates of the DCL1 primary transcript, which appears to undergo either splicing to generate full-length DCL1 mRNA or processing by DCL1 itself to generate transcript fragments severed within intron 14. (C) A schematic of DCL1 post-transcriptional autoregulation. When DCL1 protein levels are high, it could compete with splicing machinery for access to intron 14, thereby supplementing miR162-mediated regulation to maintain the proper level of DCL1 mRNA.

Figure 5.

The TAS4 locus gives rise to tasiRNAs predicted to down-regulate MYB transcripts. (A) The number of reads with a 5′ terminus at each position is plotted. Bars above the axis represent sense reads; those below represent antisense reads. The miR828 complementary site is marked by a red arrow and shown below the graph, together with the fraction of 5′ RACE clones supporting the indicated cleavage site. TAS4-siR81(−), the siRNA predicted to target MYB genes, is indicated (blue bar), as is the spacing separating the phased species at each interval; spacing for the species not represented by reads is indicated in gray. (B) TAS4-siR81(−) and miR828 complementary sites in three MYB genes. Cleavage confirmed by 5′ RACE is indicated (arrows), along with the fraction of clones mapping to the cleavage site. The remaining 14 PAP2 clones mapped >20 nt from the cleavage site.

Figure 6.

Normalized abundance of candidate siRNAs in 0.1-Mb windows spanning the nuclear genome. Colored bars above the axis represent matches to the plus strand; colored bars below the axis represent those to the minus strand, with the colors indicating the proportion of 21mers (red), 24mers (light and dark blue), 24mers with a 5′ A (light blue), and other lengths (yellow). Below the siRNA profiles are histograms plotting the fraction of nucleotides falling within annotated protein-coding genes (black; scale, 0%–100%) and the fraction falling within repetitive elements annotated by RepeatMasker (gray; scale, 0%–100%). Centromeres are indicated by solid gray bars, and heterochromatic knobs are indicated by hashed gray bars.