Endogenous and silencing-associated small RNAs in plants

Abstract

A large set of endogenous small RNAs of predominantly 21 to 24 nucleotides was identified in Arabidopsis. These small RNAs resembled micro-RNAs from animals and were similar in size to small interfering RNAs that accumulated during RNA silencing triggered by multiple types of inducers. Among the 125 sequences identified, the vast majority (90%) arose from intergenic regions, although small RNAs corresponding to predicted protein-coding genes, transposon-like sequences, and a structural RNA gene also were identified. Evidence consistent with the derivation of small RNAs of both polarities, and from highly base-paired precursors, was obtained through the identification and analysis of clusters of small RNA loci. The accumulation of specific small RNAs was regulated developmentally. We propose that Arabidopsis small RNAs participate in a wide range of post-transcriptional and epigenetic events.

Figure 1.

RNA Silencing and siRNAs in an Agrobacterium Infiltration System. (A) Constructs used in the transient assay. The 35S:GFP construct contains a functional copy of the GFP coding region, and the 35S:dsGFP construct contains both sense and antisense GFP sequences separated by an intron. The constructs were introduced into Agrobacterium and injected into N. benthamiana. (B) Silencing-associated siRNAs from Agrobacterium-inoculated tissue. Small RNA from injected tissues was analyzed with a radiolabeled GFP-specific probe. A series of two exposures (0.5 and 2 h) is shown. RNA standards of 21 and 24 nucleotides containing 5′ phosphate and 3′ hydroxyl ends were used (M). Equivalent loading of samples was shown by staining the gel with ethidium bromide before transfer (bottom gel).

Figure 2.

Cloning and Analysis of siRNAs from N. benthamiana Tissue. (A) 5′ (A1) and 3′ (A2) adapters with end modifications to ensure unidirectional ligation were ligated sequentially to gel-purified siRNAs using RNA ligase (RL), and the products were amplified by reverse transcriptase–mediated PCR. nts, nucleotides. (B) RNA gel blot of intermediate and final siRNA-adapter ligation products using a GFP probe. The A1-siRNA ligation product was gel eluted (elut) before ligation with A2. The arrow shows the final A1-siRNA-A2 product. (C) Distribution of cloned siRNA sequences in the GFP sense and antisense sequences using 35S:GFP (weak inducer) and 35S:dsGFP (strong inducer).

Figure 3.

Size of Small RNAs and Distribution of Loci with Complete Identity to Small RNAs in the Arabidopsis Genome. (A) Histogram of sizes of siRNAs produced in N. benthamiana (gray bars) and small RNAs in Arabidopsis inflorescence tissue (black bars). nt, nucleotides. (B) The 125 small RNAs isolated from Arabidopsis hit 539 genome loci. A vertical line marks each locus. Several loci had a high density of small RNA sequences, and these may appear as a single line. (C) Number of small RNA loci (gray bars) on each of the five chromosomes (black bars). (D) The Arabidopsis small RNA sequences corresponded to sequences from protein-coding genes, transposons and retroelements, structural RNA genes, and IGRs.

Figure 4.

Predicted Secondary Structures of RNA from Loci Containing Clusters of IGR-Derived Small RNA Sequences. Small RNA sequences (highlighted) occurred in both sense (blue) and antisense (orange) polarities. The orientation of the known or predicted genes (accession numbers are indicated) flanking each small RNA locus is shown at right by the large arrows (black), along with genome coordinates (nucleotides) as annotated in GenBank. The orientation of the small RNAs in relation to the chromosome is represented by the orange and blue arrows. Chr, chromosome.

Figure 5.

IGR-Derived Small RNAs in Arabidopsis Tissues. Small RNA gel blots of duplicate samples isolated from three tissue types of Arabidopsis were probed with radiolabeled antisense oligonucleotides corresponding to small RNAs 19, 96, and 5. The zone in the small RNA 5 blot above the small RNA position is presented to show that putative precursor RNAs were not detected. The position of the tRNA/5S RNA zone in the blot was determined by staining with ethidium bromide. RNA from N. benthamiana leaf tissue also was analyzed. The RNA standards (21 and 24 nucleotides) and sample loading controls are described in Figure 1.

Figure 6.

Small RNAs in and around Protein-Coding Sequences with Unusual Potential to Form Extensive Secondary Structure. (A) Predicted secondary structures for RNA encompassing four loci within a 4000-nucleotide segment of chromosome V. Two loci in opposite orientations (2.1 and 2.2) correspond to small RNA 2, one within a predicted IGR (blue) and another in sequences near the 3′ end of a protein-coding gene of unknown function (orange). The predicted open reading frame is indicated by gray shading. Chr, chromosome. (B) Blot analysis of duplicate small RNA samples isolated from three Arabidopsis tissues. Duplicate sets of small RNA samples were analyzed using radiolabeled probes to detect sense or antisense versions of small RNA 2. The RNA standards (21 and 24 nucleotides) and sample loading controls are described in Figure 1.

Figure 7.

Characterization of Small RNA 39. (A) Predicted secondary structure of the IGR region corresponding to small RNA 39 (blue) and the upstream flanking sequence. The orientation (black arrows) and genome coordinates (nucleotides as annotated in GenBank) of predicted genes flanking the IGR sequence are shown. (B) Three genes in the Scarecrow-like gene family with sequences complementary (long arrow) to that of small RNA 39. For continuity with supplemental data online, the small RNA 39 sequence is written 5′ to 3′. (C) Dendrogram of the Scarecrow-like gene family. Genes highlighted in boldface contained sequences complementary to that of small RNA 39. (D) Blot analysis of duplicate small RNA samples isolated from three tissue types of Arabidopsis using a radiolabeled oligonucleotide that was complementary to small RNA 39. The RNA standards (21 and 24 nucleotides) and sample loading controls are described in Figure 1. Chr, chromosome; nts, nucleotides.