The expanding world of small RNAs in plants

Abstract

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.

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 (pri)-miRNAs, which are processed by DICER-LIKE 1 (DCL1) into a shorter stem-loop structure called precursor (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 ). MiRNAs are involved in post-transcriptional gene silencing (PTGS) by mediating mRNA cleavage or translational repression. Longer Pol II-derived hairpins, termed hairpin-derived small-interfering RNAs (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 as proto-MIRs. Natural-antisense small-interfering RNAs (natsiRNA; right) are produced from dsRNAs originating from overlapping transcription (cis-natsiRNA) or highly complementary transcripts originated from different loci (trans-natsiRNA)^–. 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 double-stranded RNA (dsRNA) by RNA-DEPENDENT RNA POLYMERASE 6 (RDR6), and processed by DCL2 and DCL4 to produce siRNAs of 22- or 21-nucleotide (nt) in length, respectively. Secondary siRNAs are mostly involved in PTGS, but can also initiate RNA-directed DNA methylation (RdDM) at specific loci. They are subdivided into trans-acting siRNAs (tasiRNA)^,,,, phased siRNA (phasiRNA) or epigenetically-activated siRNA (easiRNAs) ^,. c. Heterochromatic siRNAs are derived from transposable elements and repeats located at pericentromeric chromatin. Their biogenesis requires Pol IV transcription and the synthesis of dsRNA by RDR2, which is subsequently processed into 24-nucleotide long siRNAs by DCL3. These small RNAs are involved in maintaining RdDM-mediated transcriptional gene silencing (TGS) (reviewed in ).

Figure 2. 2′-O-methylation, uridylation and degradation of miRNAs in A. 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 SUPRESSOR 1 (HESO1) and UTP:RNA URYDILTRANSFERASE 1 (URT1) are nucleotidyl transferases that uridylate unprotected 3′ ends of small RNAs, triggering their degradation by the 3′-5′ exonucleases SMALL RNA DEGRADING NUCLEASE (SDN; middle). ARGONAUTE 1 (AGO1) recruits HESO1 during mRNA target recognition and cleavage in order to polyuridylate and degrade the 3′ of cleaved target transcripts. Thus, the 3′ methylation of miRNAs loaded onto AGO1 serves to protect them from HESO1 activity. Recent studies have shown that URT1 also interacts with AGO1 to establish mono-uridylation of particular miRNAs^, (left), 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) (see Fig. 3). 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 preference for tailing 3′-uracil, whereas URT1 prefers 3′-adenine. 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 found in vivo^,.

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 small-interfering RNAs (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 a two-hit system utilizing two 21-nucleotide (nt) miRNAs per transcript, and requires the activity of an RNA-inducing silencing complex comprising 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 dsRNA synthesis aided by SUPPRESSOR OF GENE SILENCING 3 (SGS3), and processing of 21 and 22-nucleotide siRNAs by DICER-LIKE 4 (DCL4) and DCL2, respectively. RNA Polymerase II (Pol II)-derived transcripts might also produce miRNA-independent secondary siRNA via interactions with other RNA processing machineries such as the spliceosome, or during RNA decay^,, but these pathways are not fully understood. b. An additional phasiRNA biogenesis pathway was found in monocot plants such as maize and rice, which involves the transcription of non-coding PHAS transcripts form intergenic loci. Two miRNAs (miR2118 and miR2275) were found involved in cleavage of PHAS transcripts by an unknown AGO protein. These cleavage products are converted into dsRNA by RDR6 and SGS3, and processed into 21- and 24-nucleotide phasiRNAs by DCL4 and DCL5, respectively (reviewed in ).

Figure 4. The transition from silencing by PTGS to silencing by TGS in transgenes, epialleles and active transposons

a. Post-transcriptional gene silencing (PTGS) by miRNAs is likely the major pathway triggering 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 (see Fig. 3). 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 ARGONAUTE 6 (AGO6). This pathway is able to target nascent Pol II transcripts and recruit the DNA methyltransferase DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2) to establish DNA methylation in all sequence contexts (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 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. b. CHG (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 3 Lys 9 (H3K9me2) (2). A complete PTGS-to-TGS switch occurs when SAWADEE HOMEODOMAIN HOMOLOG 1 (SHH1) binds H3K9me2 and recruits RNA Polymerase IV (Pol IV) to initiate the biogenesis of 24-nt siRNAs through RDR2 and DCL3 (3). RdDM consolidation is achieved by the recruitment of Pol V to unmethylated DNA by SU(VAR)3–9 HOMOLOG 2 (SUVH2) and SUVH9 (4). This is followed by the recruitment of AGO4, mediated by sequence complementarity between the 24-nt siRNAs and the Pol V-nascent transcripts, and by the conserved GW/WG motif (also known as Ago hook) present in the carboxy-terminal region of the Pol V subunit NRPE1. Then AGO4 is able to recruit DRM2 to establish additional DNA methylation de novo (reviewed in ^and ).

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) phasiRNAs are produced upon cleavage of PHAS transcripts by miR2118, whereas miR2275 triggers 24-nucleotide phasiRNA biogenesis from a different subset of PHAS loci (reviewed in ). The spatiotemporal dynamics of phasiRNA biogenesis was recently described throughout anther development in maize, showing a distinct and mostly non-overlapping accumulation patterns for both phasiRNA classes, which nicely coincides with the expression of their respective miRNA triggers. 21-nucleotide phasiRNAs are essentially pre-meiotic, whereas 24-nucleotide 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 the MEIOSIS ARRESTED AT LEPTONENE1 (MEL1) protein, which is the ortholog of AGO5 in Arabidopsis thaliana. The mel1 mutants arrest during early meiotic stages, and produce dysfunctional pollen mother cells (PMCs) that appear frequently in developing anthers. b. ARGONAUTE (AGO) functions in meiosis, cell specification and chromosome segregation. (Left) In the female gametophyte, AGO104 in maize and AGO9 in A. thaliana were associated with non-cell-autonomous regulation of meiosis and germline specification, but the molecular pathways responsible for that are still unclear^,. Despite both being expressed in companion cells, AGO104 and AGO9 are involved in epigenetic silencing of transposable elements in the megaspore mother cells (MMC), perhaps through RdDM activity and mobile small RNA^,. (Right) Importantly, ago104 mutants also produce viable unreduced diploid gametes, indicating that AGO104 has a role meiotic chromosome segregation and establishing a direct link between small RNA regulation and apomixis. Top image in panel a adapted from . Right Image in panel b reproduced from (arrowheads indicate micronuclei in abnormal tetrads).