The expanding world of small RNAs in plants

Journal name:
Nature Reviews Molecular Cell Biology
Year published:
Published online


Plant genomes encode various small RNAs that function in distinct, yet overlapping, genetic and epigenetic silencing pathways. However, the abundance and diversity of small-RNA classes varies among plant species, suggesting coevolution between environmental adaptations and gene-silencing mechanisms. Biogenesis of small RNAs in plants is well understood, but we are just beginning to uncover their intricate regulation and activity. Here, we discuss the biogenesis of plant small RNAs, such as microRNAs, secondary siRNAs and heterochromatic siRNAs, and their diverse cellular and developmental functions, including in reproductive transitions, genomic imprinting and paramutation. We also discuss the diversification of small-RNA-directed silencing pathways through the expansion of RNA-dependent RNA polymerases, DICER proteins and ARGONAUTE proteins.

At a glance


  1. Main pathways for biogenesis of endogenous small RNAs in plants.
    Figure 1: Main pathways for biogenesis of endogenous small RNAs in plants.

    a | Genes encoding microRNAs (miRNAs; left) are transcribed by RNA polymerase II (Pol II) and fold into hairpin-like structures called primary miRNAs (pri-miRNAs), which are processed by DICER-LIKE 1 (DCL1) into shorter stem–loop structures called precursor miRNAs (pre-miRNAs). Pre-miRNAs are processed again by DCL1 into the mature miRNA duplex. During miRNA processing, DCL1 is assisted by several proteins (reviewed in Ref. 8). miRNAs are involved in post-transcriptional gene silencing (PTGS) by mediating mRNA cleavage or translational repression. Longer Pol II-derived hairpins, termed hairpin-derived siRNAs (hp-siRNAs; middle), might originate from inverted repeats and are originally processed by all DCLs. These hairpins might evolve into miRNAs and are often designated proto-miRNAs. Natural antisense siRNAs (natsiRNAs; right) are produced from double-stranded RNAs (dsRNAs) originating from overlapping transcription (cis-natsiRNAs), or from highly complementary transcripts originating from different loci (trans-natsiRNAs)174, 175, 176. The biogenesis and function of natsiRNAs is still largely unclear. b | The precursors of secondary siRNAs are transcribed by Pol II and may originate from non-coding loci, protein-coding genes and transposable elements. These transcripts are converted into dsRNA by RNA-DEPENDENT RNA POLYMERASE 6 (RDR6) and processed by DCL2 and DCL4 to produce siRNAs of 22 or 21 nucleotides (nt) in length, respectively. Secondary siRNAs are mostly involved in PTGS, but they can also initiate RNA-directed DNA methylation (RdDM) at specific loci. They are subdivided into trans-acting siRNAs (tasiRNAs)34, 66, 161, 177, phased siRNA (phasiRNAs)65 and epigenetically activated siRNAs (easiRNAs)77, 178. c | Heterochromatic siRNAs (hetsiRNAs) are derived from transposable elements and repeats that are preferentially located at pericentromeric chromatin. Their biogenesis requires Pol IV transcription and the synthesis of dsRNA by RDR2, which is subsequently processed into 24-nt-long siRNAs by DCL3. These small RNAs are involved in maintaining RdDM-mediated TGS (reviewed in Ref. 31).

  2. 2[prime]-O-methylation, uridylation and degradation of microRNAs (miRNAs) in Arabidopsis thaliana.
    Figure 2: 2′-O-methylation, uridylation and degradation of microRNAs (miRNAs) in Arabidopsis thaliana.

    MicroRNA (miRNA) duplexes are 2′-O-methylated at both 3′-ends by HUA ENHANCER 1 (HEN1), which protects them from uridylation and degradation (left). HEN SUPPRESSOR 1 (HESO1) and UTP:RNA URIDYLYLTRANSFERASE 1 (URT1) are nucleotidyl transferases that uridylate unprotected 3′-ends of small RNAs, triggering their degradation by the 3′–5′ exonucleases of the family SMALL RNA DEGRADING NUCLEASEs (SDNs; middle). ARGONAUTE 1 (AGO1) recruits HESO1 during mRNA-target recognition and cleavage to polyuridylate and degrade the 3′-end of cleaved target transcripts52. Thus, the 3′-methylation of miRNAs loaded onto AGO1 protects them from HESO1 activity. Recent studies have shown that URT1 also interacts with AGO1 to establish monouridylation of particular miRNAs53, 54 (right), and this process may produce 22-nucleotide miRNA variants that are able to form functional RNA-induced silencing complexes and trigger post-transcriptional gene silencing (PTGS)54 (Fig. 3a). HESO1 and URT1 have been shown to act both independently and synergistically, perhaps reflecting their different affinities for 3′-terminal nucleotides in vitro. HESO1 has a preference for tailing 3′-uracil, whereas URT1 prefers 3′-adenine54. Although these features explain how these enzymes act synergistically at non-3′-uracil miRNA targets (URT1 forms substrates for HESO1), it does not fully account for their substrate preferences in vivo53, 54.

  3. Triggers of secondary siRNA biogenesis.
    Figure 3: Triggers of secondary siRNA biogenesis.

    a | Plant microRNAs (miRNAs) target transcripts for cleavage or translational repression and also trigger the production of secondary siRNAs from mRNAs, non-coding RNAs and transposable elements. The most accepted mechanism for the biogenesis of trans-acting siRNA (tasiRNA), phased siRNA (phasiRNA) and epigenetically activated siRNA (easiRNA) relies on two distinct pathways. One consists of a 'two-hit' system, which uses two 21-nucleotide (nt) miRNAs per transcript and requires the activity of an RNA-induced silencing complex containing ARGONAUTE 7 (AGO7). The second pathway consists of a 'one-hit' system that usually involves a 22-nt miRNA loaded on AGO1, or 22-nt miRNA variants that are produced from monouridylation of 21-nt miRNAs (see Fig. 2). Both pathways are routed towards RNA-DEPENDENT RNA POLYMERASE 6 (RDR6)-mediated double-stranded RNA (dsRNA) synthesis, aided by SUPPRESSOR OF GENE SILENCING 3 (SGS3), and processing of 21-nt and 22-nt siRNAs by DICER-LIKE 4 (DCL4) and DCL2, respectively. RNA polymerase II (Pol II)-derived transcripts might also produce miRNA-independent secondary siRNA through interactions with other RNA-processing machineries, such as the spliceosome85, or during RNA decay100, 101, but these pathways are not fully understood. b | An additional phasiRNA biogenesis pathway was found in monocot plants, such as maize and rice, and involves the transcription of non-coding PHAS transcripts from intergenic loci. Two miRNAs (miR2118 and miR2275) were found to be involved in cleavage of PHAS transcripts by an unknown AGO. These cleavage products are converted into dsRNA by RDR6 and SGS3, and processed into 21- and 24-nt phasiRNAs by DCL4 and DCL5, respectively (reviewed in Ref. 65).

  4. The transition from post-transcriptional gene silencing (PTGS) to TGS in transgenes, epialleles and active transposons.
    Figure 4: The transition from post-transcriptional gene silencing (PTGS) to TGS in transgenes, epialleles and active transposons.

    a | PTGS by microRNAs (miRNAs) is probably the major pathway triggering the biogenesis of secondary 21- and 22-nucleotide (nt) siRNAs, in a process involving RNA-DEPENDENT RNA POLYMERASE 6 (RDR6), SUPPRESSOR OF GENE SILENCING 3 (SGS3), DICER-LIKE 4 (DCL4) and DCL2 (Fig. 3). These 21- and 22-nt siRNAs are required for the establishment of RNA-directed DNA methylation (RdDM) at particular transposable elements and epialleles, which (at least at some loci) requires the activity of ARGONAUTE 6 (AGO6)109. This pathway is able to target nascent RNA polymerase II (Pol II) transcripts and recruit the DNA methyltransferase DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2) to establish DNA methylation in all sequence contexts (step 1), but this interplay is not fully understood. An alternative pathway was proposed for transgenes and active retrotransposons, perhaps depending on their variable copy number and transcription levels. The accumulation of long double-stranded RNA (dsRNA) molecules might saturate both the DCL2- and DCL4-processing pathways, resulting in functional compensation by DCL3, which instead produces 24-nt siRNAs for the establishment of RdDM via AGO4 (Ref. 110). b | CHG (where H denotes A, C or T) methylation, previously established by DRM2, is recognized by the histone methyltransferase KRYPTONITE (KYP), which reinforces the repressed chromatin state of methylated DNA by establishing the dimethylation of histone H3 at Lys9 (H3K9me2)121 (step 2). A complete PTGS-to-TGS switch occurs when SAWADEE HOMEODOMAIN HOMOLOGUE 1 (SHH1) binds to H3K9me2 and recruits Pol IV to initiate the biogenesis of 24-nt siRNAs through RDR2 and DCL3 (Ref. 122) (step 3). RdDM consolidation is achieved by the recruitment of Pol V to methylated DNA by SU(VAR)3-9 HOMOLOGUE 2 (SUVH2) or SUVH9 (Ref. 123) (step 4). This is followed by the recruitment of AGO4, mediated by sequence complementarity between the 24-nt siRNAs and the nascent Pol V transcripts, and by the conserved GW/WG motif (also known as the AGO hook) present in the carboxy-terminal region of the Pol V subunit NRPE1. AGO4 is then able to recruit DRM2 to establish additional DNA methylation de novo (reviewed in Refs 31, 112).

  5. Small-RNA functions in meiosis and cell fate specification.
    Figure 5: Small-RNA functions in meiosis and cell fate specification.

    a | In grass anthers, two distinct small-RNA classes are produced from non-coding PHAS transcripts: 21-nucleotide (nt) phased siRNAs (phasiRNAs) are produced on cleavage of PHAS transcripts by miR2118, whereas 24-nt phasiRNAs are produced from a different subset of PHAS loci after triggering by miR2275 (reviewed in Ref. 65). The spatiotemporal dynamics of phasiRNA biogenesis was recently described throughout anther development in maize72, showing distinct and mostly non-overlapping accumulation patterns for both phasiRNA classes, which coincide with the expression of their respective microRNA (miRNA) triggers. 21-nt phasiRNAs are essentially pre-meiotic, whereas 24-nt phasiRNAs peak during meiosis and decrease during pollen development. The function of these male-specific small RNAs remains unknown, but their different size and accumulation patterns suggest distinct biological activities. A subset of 21-nt phasiRNAs in rice is loaded onto MEIOSIS ARRESTED AT LEPTONENE 1 (MEL1)150, which is the orthologue of ARGONAUTE 5 (AGO5) in Arabidopsis thaliana. mel1 mutants arrest during early meiotic stages and produce dysfunctional pollen mother cells (PMCs) that appear frequently in developing anthers. b | AGO functions in meiosis, cell specification and chromosome segregation. In the female gametophyte (left), AGO104 in maize and AGO9 in A. thaliana are associated with non-cell-autonomous regulation of meiosis and germline specification, but the molecular pathways responsible are still unclear147, 149. Despite both being expressed in companion cells, AGO104 and AGO9 are involved in epigenetic silencing of transposable elements (TEs) in the megaspore mother cells (MMCs), perhaps through RNA-directed DNA methylation (RdDM) activity and mobile small RNAs147, 149. Importantly, ago104 mutants also produce viable unreduced diploid gametes (right; arrowheads indicate micronuclei in abnormal tetrads), indicating that AGO104 has a role in meiotic chromosome segregation and in establishing a direct link between small-RNA regulation and apomixis147. WT, wild type. Top image in part a adapted with permission from Ref. 72, National Academy of Sciences. Right image in part b from Ref. 147. The plant cell by American Society of Plant Physiologists. Reproduced with permission of AMERICAN SOCIETY OF PLANT PHYSIOLOGISTS in the format Republish in a journal/magazine via Copyright Clearance Center.


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  1. Howard Hughes Medical Institute and Gordon and Betty Moore Foundation, Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, New York 11724, USA.

    • Filipe Borges &
    • Robert A. Martienssen

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  • Filipe Borges

    Filipe Borges is a postdoctoral fellow in Rob Martienssen's laboratory at Cold Spring Harbor Laboratory, New York, USA. He is studying reprogramming and transgenerational silencing of transposable elements in pollen by single-cell genomics and epigenomics, with a focus on the evolution of epigenetic regulation in eukaryotic systems. His training and graduate research at the Instituto Gulbenkian de Ciência in Oeiras, Portugal, focused on understanding the molecular mechanisms regulating germline specification in Arabidopsis thaliana and on developing new tools for the purification of plant cells by fluorescence-activated cell sorting.

  • Robert A. Martienssen

    Robert A. Martienssen leads the plant biology group at Cold Spring Harbor Laboratory, New York, USA, where he focuses on epigenetic mechanisms that shape and regulate the genome and their effect on development and inheritance. His work on transposons, or 'jumping genes', in plants and in fission yeast revealed a link between heterochromatin and RNAi. His laboratory currently focuses on mechanistic aspects of germline reprogramming and epigenetic inheritance, including DNA methylation, histone replacement and modification, and RNAi.

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