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doi: 10.1261/rna.1052008. Epub 2008 Jun 19.

Conifers have a unique small RNA silencing signature

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Conifers have a unique small RNA silencing signature

Elena V Dolgosheina et al. RNA.2008 Aug.

Abstract

Plants produce small RNAs to negatively regulate genes, viral nucleic acids, and repetitive elements at either the transcriptional or post-transcriptional level in a process that is referred to as RNA silencing. While RNA silencing has been extensively studied across the different phyla of the animal kingdom (e.g., mouse, fly, worm), similar studies in the plant kingdom have focused primarily on angiosperms, thus limiting evolutionary studies of RNA silencing in plants. Here we report on an unexpected phylogenetic difference in the size distribution of small RNAs among the vascular plants. By extracting total RNA from freshly growing shoot tissue, we conducted a survey of small RNAs in 24 vascular plant species. We find that conifers, which radiated from the other seed-bearing plants approximately 260 million years ago, fail to produce significant amounts of 24-nucleotide (nt) RNAs that are known to guide DNA methylation and heterochromatin formation in angiosperms. Instead, they synthesize a diverse population of small RNAs that are exactly 21-nt long. This finding was confirmed by high-throughput sequencing of the small RNA sequences from a conifer, Pinus contorta. A conifer EST search revealed the presence of a novel Dicer-like (DCL) family, which may be responsible for the observed change in small RNA expression. No evidence for DCL3, an enzyme that matures 24-nt RNAs in angiosperms, was found. We hypothesize that the diverse class of 21-nt RNAs found in conifers may help to maintain organization of their unusually large genomes.

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Figures

FIGURE 1.
FIGURE 1.
Conifers are notable in that they lack a detectable 24-nt population of small RNAs, otherwise found in a diverse range of vascular plants, while producing a dominant 21-nt class of small RNAs. Polyacrylamide gel analysis was performed to investigate total RNA size profiles from 24 vascular plant species. Pteridophytes: (lanes1–3) clubmoss, horsetail, quillwort, and (lanes4,5) ferns. (Lanes6–12) Conifer gymnosperms: Western red cedar, monkey-puzzle tree, Norway spruce, Eastern white pine, white spruce, Douglas-fir, and lodgepole pine. (Lanes13–15) Other gymnosperms: ephedra, cycad, and ginkgo. (Lane16) Ancient angiosperms: water lily. (Lanes17–19) Monocot angiosperms: barley, maize, and rice. (Lanes20–24) Eudicot angiosperms: lettuce, pea, cauliflower, tobacco, and tomato. (M) Marker lane. Small RNAs from plant species marked with an asterisk were subjected to high throughput 454 sequencing. The phylogeny of major vascular plant divisions is shown below the gel image (Schneider et al. 2004).
FIGURE 2.
FIGURE 2.
Length distribution of total small RNA and conserved miRNA sequences obtained fromO. sativa andP. contorta. (A) Small RNAs fromO. sativa exhibit the profile typical of an angiosperm sample having a dominant 24-nt RNA population together with a smaller fraction of 21–22-nt RNA. (B) In contrast, small RNAs fromP. contorta were dominated by a 21-nt class of small RNAs. This class was very diverse in sequence composition. (C) Detection of known miRNAs. Conserved miRNAs fromP. contorta (red bars) andO. sativa (blue bars) were identified by sequence similarity comparisons with known miRNA sequences deposited in miRBase release 10.0 (http://microrna.sanger.ac.uk).O. sativa: 99 distinct miRNA sequences were found corresponding to 18 known miRNA families (i.e., having a unique random sequence tag).P. contorta: 173 distinct miRNA sequences were found corresponding to 22 known miRNA families. (D) Fractions of known miRNAs are plotted by size; (red bars)P. contorta; (blue bars)O. sativa. Data adapted from Morin et al. (2008).
FIGURE 3.
FIGURE 3.
Sodium periodate treatment of small RNAs fromCyrtomium sp. (fern),P. contorta (lodgepole pine), andBrassica oleracea (cauliflower). Only if both the 2′- and 3′-hydroxyl groups are unmodified will β-elimination of the terminal nucleoside take place, resulting in an ∼2-nt increase in electrophoretic mobility. Small RNAs in the 21–24-nt range for all three plant samples were highly resistant to the sodium periodate treatment, as exemplified by the comigration of small RNA bands in the − and + periodate lanes. A synthetic 11-nt RNA 5′-end-labeled with32P was added as an internal positive control (lower band) to all RNA extracts before chemical treatment to confirm the efficient periodate-mediated elimination of the terminal nucleoside from unmodified RNA. The size marker on theleft is 24-nt long, the marker on theright is 21-nt long.
FIGURE 4.
FIGURE 4.
Identification of a new Dicer-like family in conifers. (A) Domain organization of a flowering plant Dicer. Dicers inA. thaliana andO. sativa have a total of eight domains with the following exceptions: DCL2 lacks second dsRBD; DCL3 lacks Duf283 (Margis et al. 2006). (B) Amino acid sequences corresponding to seven DCL-like ESTs fromP. taeda andPicea spp., and the C-terminal regions from fourA. thaliana Dicers, sixO. sativa Dicers, and two predicted Dicers fromM. truncatula were aligned using ClustalX and default parameters. Only the alignment of the region exhibiting the highest sequence conservation is shown (90 amino acids). This region is predicted to correspond to the end of the second RNase III domain and to part of the dsRBD. Color code for amino acid residues: (orange) GPST; (red) HKR; (blue) FWY; (green) ILMV. Bars below the alignment indicate prevalence of the most common amino acid residue at each position, with the highest bar corresponding to 100% conservation. (C) Phylogenetic tree of Dicers in the seed-bearing plants generated from the alignment inB. Positions with gaps were excluded during tree building.
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