The expanding world of small RNAs in plants

Journal name:
Nature Reviews Molecular Cell Biology
Volume:
16,
Pages:
727–741
Year published:
DOI:
doi:10.1038/nrm4085
Published online

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.

At a glance

Figures

  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.

References

  1. Axtell, M. J. Classification and comparison of small RNAs from plants. Annu. Rev. Plant Biol. 64, 137159 (2013).
  2. Henderson, I. R. et al. Dissecting Arabidopsis thaliana DICER function in small RNA processing, gene silencing and DNA methylation patterning. Nat. Genet. 38, 721725 (2006).
  3. Gasciolli, V., Mallory, A. C., Bartel, D. P. & Vaucheret, H. Partially redundant functions of Arabidopsis DICER-like enzymes and a role for DCL4 in producing trans-acting siRNAs. Curr. Biol. 15, 14941500 (2005).
  4. Mukherjee, K., Campos, H. & Kolaczkowski, B. Evolution of animal and plant Dicers: early parallel duplications and recurrent adaptation of antiviral RNA binding in plants. Mol. Biol. Evol. 30, 627641 (2013).
  5. Willmann, M. R., Endres, M. W., Cook, R. T. & Gregory, B. D. The functions of RNA-dependent RNA polymerases in Arabidopsis. Arabidopsis Book 9, e0146 (2011).
  6. Czech, B. & Hannon, G. J. Small RNA sorting: matchmaking for Argonautes. Nat. Rev. Genet. 12, 1931 (2010).
  7. Weinberg, D. E., Nakanishi, K., Patel, D. J. & Bartel, D. P. The inside-out mechanism of Dicers from budding yeasts. Cell 146, 262276 (2011).
  8. Bologna, N. G. & Voinnet, O. The diversity, biogenesis, and activities of endogenous silencing small RNAs in Arabidopsis. Annu. Rev. Plant Biol. 65, 473503 (2014).
  9. Nodine, M. D. & Bartel, D. P. MicroRNAs prevent precocious gene expression and enable pattern formation during plant embryogenesis. Genes Dev. 24, 26782692 (2010).
    Demonstrates that the miRNA miR156 plays an essential part in the early development of A. thaliana embryos.
  10. Schauer, S. E., Jacobsen, S. E., Meinke, D. W. & Ray, A. DICER-LIKE1: blind men and elephants in Arabidopsis development. Trends Plant Sci. 7, 487491 (2002).
  11. Liu, B. et al. Loss of function of OsDCL1 affects microRNA accumulation and causes developmental defects in rice. Plant Physiol. 139, 296305 (2005).
  12. Thompson, B. E. et al. The dicer-like1 homolog fuzzy tassel is required for the regulation of meristem determinacy in the inflorescence and vegetative growth in maize. Plant Cell 26, 47024717 (2014).
  13. Cuperus, J. T., Fahlgren, N. & Carrington, J. C. Evolution and functional diversification of MIRNA genes. Plant Cell 23, 431442 (2011).
  14. Fahlgren, N. et al. High-throughput sequencing of Arabidopsis microRNAs: evidence for frequent birth and death of MIRNA genes. PLoS ONE 2, e219 (2007).
  15. Chellappan, P. et al. siRNAs from miRNA sites mediate DNA methylation of target genes. Nucleic Acids Res. 38, 68836894 (2010).
  16. Vazquez, F., Blevins, T., Ailhas, J., Boller, T. & Meins, F. Evolution of Arabidopsis MIR genes generates novel microRNA classes. Nucleic Acids Res. 36, 64296438 (2008).
  17. Wu, L. et al. DNA methylation mediated by a microRNA pathway. Mol. Cell 38, 465475 (2010).
  18. Rajagopalan, R., Vaucheret, H., Trejo, J. & Bartel, D. P. A diverse and evolutionarily fluid set of microRNAs in Arabidopsis thaliana. Genes Dev. 20, 34073425 (2006).
  19. Ben Amor, B. et al. Novel long non-protein coding RNAs involved in Arabidopsis differentiation and stress responses. Genome Res. 19, 5769 (2009).
  20. Allen, E. et al. Evolution of microRNA genes by inverted duplication of target gene sequences in Arabidopsis thaliana. Nat. Genet. 36, 12821290 (2004).
  21. Fahlgren, N. et al. MicroRNA gene evolution in Arabidopsis lyrata and Arabidopsis thaliana. Plant Cell 22, 10741089 (2010).
  22. Axtell, M. J., Westholm, J. O. & Lai, E. C. Vive la différence: biogenesis and evolution of microRNAs in plants and animals. Genome Biol. 12, 221 (2011).
  23. Piriyapongsa, J. & Jordan, I. K. Dual coding of siRNAs and miRNAs by plant transposable elements. RNA 14, 814821 (2008).
  24. Zhang, Y., Jiang, W.-K. & Gao, L.-Z. Evolution of microRNA genes in Oryza sativa and Arabidopsis thaliana: an update of the inverted duplication model. PLoS ONE 6, e28073 (2011).
  25. Tang, G., Reinhart, B. J., Bartel, D. P. & Zamore, P. D. A biochemical framework for RNA silencing in plants. Genes Dev. 17, 4963 (2003).
  26. Moissiard, G., Parizotto, E. A., Himber, C. & Voinnet, O. Transitivity in Arabidopsis can be primed, requires the redundant action of the antiviral Dicer-like 4 and Dicer-like 2, and is compromised by viral-encoded suppressor proteins. RNA 13, 12681278 (2007).
  27. Zong, J., Yao, X., Yin, J., Zhang, D. & Ma, H. Evolution of the RNA-dependent RNA polymerase (RdRP) genes: duplications and possible losses before and after the divergence of major eukaryotic groups. Gene 447, 2939 (2009).
  28. Garcia-Ruiz, H. et al. Arabidopsis RNA-dependent RNA polymerases and dicer-like proteins in antiviral defense and small interfering RNA biogenesis during Turnip Mosaic Virus infection. Plant Cell 22, 481496 (2010).
  29. Castel, S. E. & Martienssen, R. A. RNA interference in the nucleus: roles for small RNAs in transcription, epigenetics and beyond. Nat. Rev. Genet. 14, 100112 (2013).
  30. Slotkin, R. K. & Martienssen, R. Transposable elements and the epigenetic regulation of the genome. Nat. Rev. Genet. 8, 272285 (2007).
  31. Matzke, M. A. & Mosher, R. A. RNA-directed DNA methylation: an epigenetic pathway of increasing complexity. Nat. Rev. Genet. 15, 394408 (2014).
  32. Parent, J.-S., Bouteiller, N., Elmayan, T. & Vaucheret, H. Respective contributions of Arabidopsis DCL2 and DCL4 to RNA silencing. Plant J. 81, 223232 (2015).
  33. Wang, X.-B. et al. RNAi-mediated viral immunity requires amplification of virus-derived siRNAs in Arabidopsis thaliana. Proc. Natl Acad. Sci. USA 107, 484489 (2010).
  34. Yoshikawa, M., Peragine, A., Park, M.-Y. & Poethig, R. S. A pathway for the biogenesis of trans-acting siRNAs in Arabidopsis. Genes Dev. 19, 21642175 (2005).
  35. Hiraguri, A. et al. Specific interactions between Dicer-like proteins and HYL1/DRB-family dsRNA-binding proteins in Arabidopsis thaliana. Plant Mol. Biol. 57, 173188 (2005).
  36. Kim, Y.-K., Heo, I. & Kim, V. N. Modifications of small RNAs and their associated proteins. Cell 143, 703709 (2010).
  37. Li, J., Yang, Z., Yu, B., Liu, J. & Chen, X. Methylation protects miRNAs and siRNAs from a 3′-end uridylation activity in Arabidopsis. Curr. Biol. 15, 15011507 (2005).
  38. Zhai, J. et al. Plant microRNAs display differential 3′ truncation and tailing modifications that are ARGONAUTE1 dependent and conserved across species. Plant Cell 25, 24172428 (2013).
    Addresses the prevalence, conservation and biological significance of truncated and uridylated miRNA variants in plants.
  39. Yu, B. et al. Methylation as a crucial step in plant microRNA biogenesis. Science 307, 932935 (2005).
  40. Zhao, Y. et al. The Arabidopsis nucleotidyl transferase HESO1 uridylates unmethylated small RNAs to trigger their degradation. Curr. Biol. 22, 689694 (2012).
  41. Ren, G., Chen, X. & Yu, B. Uridylation of miRNAs by HEN1 SUPPRESSOR1 in Arabidopsis. Curr. Biol. 22, 695700 (2012).
  42. Ramachandran, V. & Chen, X. Degradation of microRNAs by a family of exoribonucleases in Arabidopsis. Science 321, 14901492 (2008).
  43. Chen, X., Liu, J., Cheng, Y. & Jia, D. HEN1 functions pleiotropically in Arabidopsis development and acts in C function in the flower. Development 129, 10851094 (2002).
  44. Ibrahim, F. et al. Uridylation of mature miRNAs and siRNAs by the MUT68 nucleotidyltransferase promotes their degradation in Chlamydomonas. Proc. Natl Acad. Sci. USA 107, 39063911 (2010).
  45. Wyman, S. K. et al. Post-transcriptional generation of miRNA variants by multiple nucleotidyl transferases contributes to miRNA transcriptome complexity. Genome Res. 21, 14501461 (2011).
  46. Hagan, J. P., Piskounova, E. & Gregory, R. I. Lin28 recruits the TUTase Zcchc11 to inhibit let-7 maturation in mouse embryonic stem cells. Nat. Struct. Mol. Biol. 16, 10211025 (2009).
  47. Heo, I. et al. TUT4 in concert with Lin28 suppresses microRNA biogenesis through pre-microRNA uridylation. Cell 138, 696708 (2009).
  48. Heo, I. et al. Mono-uridylation of pre-microRNA as a key step in the biogenesis of group II let-7 microRNAs. Cell 151, 521532 (2012).
  49. van Wolfswinkel, J. C. et al. CDE-1 affects chromosome segregation through uridylation of CSR-1-bound siRNAs. Cell 139, 135148 (2009).
  50. Shirayama, M. et al. piRNAs initiate an epigenetic memory of nonself RNA in the C. elegans germline. Cell 150, 6577 (2012).
  51. Wedeles, C. J., Wu, M. Z. & Claycomb, J. M. Protection of germline gene expression by the C. elegans Argonaute CSR-1. Dev. Cell 27, 664671 (2013).
  52. Ren, G. et al. Methylation protects microRNAs from an AGO1-associated activity that uridylates 5′ RNA fragments generated by AGO1 cleavage. Proc. Natl Acad. Sci. USA 111, 63656370 (2014).
  53. Wang, X. et al. Synergistic and independent actions of multiple terminal nucleotidyl transferases in the 3′ tailing of small RNAs in Arabidopsis. PLoS Genet. 11, e1005091 (2015).
  54. Tu, B. et al. Distinct and cooperative activities of HESO1 and URT1 nucleotidyl transferases in microRNA turnover in Arabidopsis. PLoS Genet. 11, e1005119 (2015).
  55. Ryvkin, P. et al. HAMR: high-throughput annotation of modified ribonucleotides. RNA 19, 16841692 (2013).
  56. Ebhardt, H. A. et al. Meta-analysis of small RNA-sequencing errors reveals ubiquitous post-transcriptional RNA modifications. Nucleic Acids Res. 37, 24612470 (2009).
  57. Iida, K., Jin, H. & Zhu, J.-K. Bioinformatics analysis suggests base modifications of tRNAs and miRNAs in Arabidopsis thaliana. BMC Genomics 10, 155 (2009).
  58. Yan, J., Zhang, H., Zheng, Y. & Ding, Y. Comparative expression profiling of miRNAs between the cytoplasmic male sterile line MeixiangA and its maintainer line MeixiangB during rice anther development. Planta 241, 109123 (2015).
  59. Kierzek, E. et al. The contribution of pseudouridine to stabilities and structure of RNAs. Nucleic Acids Res. 42, 34923501 (2014).
  60. Carlile, T. M. et al. Pseudouridine profiling reveals regulated mRNA pseudouridylation in yeast and human cells. Nature 515, 143146 (2014).
  61. Schwartz, S. et al. Transcriptome-wide mapping reveals widespread dynamic-regulated pseudouridylation of ncRNA and mRNA. Cell 159, 148162 (2014).
  62. Simos, G. et al. The yeast protein Arc1p binds to tRNA and functions as a cofactor for the methionyl- and glutamyl-tRNA synthetases. EMBO J. 15, 54375448 (1996).
  63. Hellmuth, K. et al. Cloning and characterization of the Schizosaccharomyces pombe tRNA:pseudouridine synthase Pus1p. Nucleic Acids Res. 28, 46044610 (2000).
  64. Zhai, J. et al. MicroRNAs as master regulators of the plant NB-LRR defense gene family via the production of phased, trans-acting siRNAs. Genes Dev. 25, 25402553 (2011).
  65. Fei, Q., Xia, R. & Meyers, B. C. Phased, secondary, small interfering RNAs in posttranscriptional regulatory networks. Plant Cell 25, 24002415 (2013).
  66. Allen, E., Xie, Z., Gustafson, A. M. & Carrington, J. C. microRNA-directed phasing during trans-acting siRNA biogenesis in plants. Cell 121, 207221 (2005).
  67. Axtell, M. J., Jan, C., Rajagopalan, R. & Bartel, D. P. A. Two-hit trigger for siRNA biogenesis in plants. Cell 127, 565577 (2006).
  68. Manavella, P. A., Koenig, D. & Weigel, D. Plant secondary siRNA production determined by microRNA-duplex structure. Proc. Natl Acad. Sci. USA 109, 24612466 (2012).
  69. Ronemus, M., Vaughn, M. W. & Martienssen, R. A. MicroRNA-targeted and small interfering RNA-mediated mRNA degradation is regulated by argonaute, dicer, and RNA-dependent RNA polymerase in Arabidopsis. Plant Cell 18, 15591574 (2006).
  70. Johnson, C. et al. Clusters and superclusters of phased small RNAs in the developing inflorescence of rice. Genome Res. 19, 14291440 (2009).
  71. Arikit, S., Zhai, J. & Meyers, B. C. Biogenesis and function of rice small RNAs from non-coding RNA precursors. Curr. Opin. Plant Biol. 16, 170179 (2013).
  72. Zhai, J. et al. Spatiotemporally dynamic, cell-type-dependent premeiotic and meiotic phasiRNAs in maize anthers. Proc. Natl Acad. Sci. USA 112, 31463151 (2015).
    Provides comprehensive spatiotemporal characterization of the biogenesis pathways and dynamics of two functionally uncharacterized, male-specific phasiRNA clusters in maize.
  73. Song, X. et al. Rice RNA-dependent RNA polymerase 6 acts in small RNA biogenesis and spikelet development. Plant J. 71, 378389 (2012).
  74. Song, X. et al. Roles of DCL4 and DCL3b in rice phased small RNA biogenesis. Plant J. 69, 462474 (2012).
  75. Shivaprasad, P. V. et al. A microRNA superfamily regulates nucleotide binding site-leucine-rich repeats and other mRNAs. Plant Cell 24, 859874 (2012).
  76. Arikit, S. et al. An atlas of soybean small RNAs identifies phased siRNAs from hundreds of coding genes. Plant Cell 26, 45844601 (2014).
  77. Creasey, K. M. et al. miRNAs trigger widespread epigenetically activated siRNAs from transposons in Arabidopsis. Nature 508, 411415 (2014).
    Shows that secondary siRNAs are produced from epigenetically activated transposable elements and that miRNAs are involved in this process.
  78. Slotkin, R. K. et al. Epigenetic reprogramming and small RNA silencing of transposable elements in pollen. Cell 136, 461472 (2009).
    Demonstrates that transposons are expressed and active in the vegetative nuclei of pollen, producing secondary siRNAs that accumulate in the neighbouring gametes and potentially reinforcing transgenerational epigenetic silencing of transposons.
  79. Tanurdzic, M. et al. Epigenomic consequences of immortalized plant cell suspension culture. PLoS Biol. 6, e302 (2008).
  80. Ito, H. et al. An siRNA pathway prevents transgenerational retrotransposition in plants subjected to stress. Nature 472, 115119 (2011).
  81. May, B. P., Lippman, Z. B., Fang, Y., Spector, D. L. & Martienssen, R. A. Differential regulation of strand-specific transcripts from Arabidopsis centromeric satellite repeats. PLoS Genet. 1, e79 (2005).
  82. Roberts, J. T., Cardin, S. E. & Borchert, G. M. Burgeoning evidence indicates that microRNAs were initially formed from transposable element sequences. Mob. Genet. Elements 4, e29255 (2014).
  83. Dumesic, P. A. et al. Stalled spliceosomes are a signal for RNAi-mediated genome defense. Cell 152, 957968 (2013).
  84. Zhang, Z. et al. The HP1 homolog rhino anchors a nuclear complex that suppresses piRNA precursor splicing. Cell 157, 13531363 (2014).
  85. Christie, M., Croft, L. J. & Carroll, B. J. Intron splicing suppresses RNA silencing in Arabidopsis. Plant J. 68, 159167 (2011).
  86. Laubinger, S. et al. Dual roles of the nuclear cap-binding complex and SERRATE in pre-mRNA splicing and microRNA processing in Arabidopsis thaliana. Proc. Natl Acad. Sci. USA 105, 87958800 (2008).
  87. Christie, M. & Carroll, B. J. SERRATE is required for intron suppression of RNA silencing in Arabidopsis. Plant Signal. Behav. 6, 20352037 (2011).
  88. Raczynska, K. D. et al. The SERRATE protein is involved in alternative splicing in Arabidopsis thaliana. Nucleic Acids Res. 42, 12241244 (2014).
  89. Ausin, I., Greenberg, M. V., Li, C. F. & Jacobsen, S. E. The splicing factor SR45 affects the RNA-directed DNA methylation pathway in Arabidopsis. Epigenetics 7, 2933 (2012).
  90. Li, S. et al. Detection of Pol IV/RDR2-dependent transcripts at the genomic scale in Arabidopsis reveals features and regulation of siRNA biogenesis. Genome Res. 25, 235245 (2015).
  91. Bayne, E. H. et al. Splicing factors facilitate RNAi-directed silencing in fission yeast. Science 322, 602606 (2008).
  92. Bernard, P., Drogat, J., Dheur, S., Genier, S. & Javerzat, J.-P. Splicing factor Spf30 assists exosome-mediated gene silencing in fission yeast. Mol. Cell. Biol. 30, 11451157 (2010).
  93. Chinen, M., Morita, M., Fukumura, K. & Tani, T. Involvement of the spliceosomal U4 small nuclear RNA in heterochromatic gene silencing at fission yeast centromeres. J. Biol. Chem. 285, 56305638 (2010).
  94. Kallgren, S. P. et al. The proper splicing of RNAi factors is critical for pericentric heterochromatin assembly in fission yeast. PLoS Genet. 10, e1004334 (2014).
  95. Gy, I. et al. Arabidopsis FIERY1, XRN2, and XRN3 are endogenous RNA silencing suppressors. Plant Cell 19, 34513461 (2007).
  96. Gazzani, S., Lawrenson, T., Woodward, C., Headon, D. & Sablowski, R. A link between mRNA turnover and RNA interference in Arabidopsis. Science 306, 10461048 (2004).
  97. Moreno, A. B. et al. Cytoplasmic and nuclear quality control and turnover of single-stranded RNA modulate post-transcriptional gene silencing in plants. Nucleic Acids Res. 41, 46994708 (2013).
  98. Thran, M., Link, K. & Sonnewald, U. The Arabidopsis DCP2 gene is required for proper mRNA turnover and prevents transgene silencing in Arabidopsis. Plant J. 72, 368377 (2012).
  99. Gregory, B. D. et al. A link between RNA metabolism and silencing affecting Arabidopsis development. Dev. Cell 14, 854866 (2008).
  100. Zhang, X. et al. Suppression of endogenous gene silencing by bidirectional cytoplasmic RNA decay in Arabidopsis. Science 348, 120123 (2015).
  101. Martínez de Alba, A. E. et al. In plants, decapping prevents RDR6-dependent production of small interfering RNAs from endogenous mRNAs. Nucleic Acids Res. 43, 29022913 (2015).
  102. Bühler, M., Haas, W., Gygi, S. P. & Moazed, D. RNAi-dependent and -independent RNA turnover mechanisms contribute to heterochromatic gene silencing. Cell 129, 707721 (2007).
  103. Yamanaka, S. et al. RNAi triggered by specialized machinery silences developmental genes and retrotransposons. Nature 493, 557560 (2013).
  104. Cao, M. et al. Virus infection triggers widespread silencing of host genes by a distinct class of endogenous siRNAs in Arabidopsis. Proc. Natl Acad. Sci. USA 111, 1461314618 (2014).
  105. Wang, X. B. et al. The 21-nucleotide, but not 22-nucleotide, viral secondary small interfering RNAs direct potent antiviral defense by two cooperative Argonautes in Arabidopsis thaliana. Plant Cell 23, 16251638 (2011).
  106. Kumakura, N. et al. SGS3 and RDR6 interact and colocalize in cytoplasmic SGS3/RDR6-bodies. FEBS Lett. 583, 12611266 (2009).
  107. McClintock, B. The suppressor-mutator system of control of gene action in maize. Carnegie Inst. Washington Year Book 57, 415429 (1958).
  108. Nuthikattu, S. et al. The initiation of epigenetic silencing of active transposable elements is triggered by RDR6 and 21–22 nucleotide small interfering RNAs. Plant Physiol. 162, 116131 (2013).
  109. McCue, A. D. et al. ARGONAUTE 6 bridges transposable element mRNA-derived siRNAs to the establishment of DNA methylation. EMBO J. 34, 2035 (2015).
  110. Mari-Ordonez, A. et al. Reconstructing de novo silencing of an active plant retrotransposon. Nat. Genet. 45, 10291039 (2013).
    Describes a study of proliferation dynamics and molecular pathways involved in restoring transcriptional silencing of a functionally active retrotransposon in A. thaliana.
  111. Havecker, E. R. et al. The Arabidopsis RNA-directed DNA methylation Argonautes functionally diverge based on their expression and interaction with target loci. Plant Cell 22, 321334 (2010).
    Shows that the functional diversification of AGO4, AGO6 and AGO9 and their preference for certain RdDM-mediating siRNAs relies on cell- and tissue-specific gene expression patterns.
  112. Law, J. A. & Jacobsen, S. E. Establishing, maintaining and modifying DNA methylation patterns in plants and animals. Nat. Rev. Genet. 11, 204220 (2010).
  113. Haag, J. R. & Pikaard, C. S. Multisubunit RNA polymerases IV and V: purveyors of non-coding RNA for plant gene silencing. Nat. Rev. Mol. Cell Biol. 12, 483492 (2011).
  114. Zheng, X., Zhu, J., Kapoor, A. & Zhu, J.-K. Role of Arabidopsis AGO6 in siRNA accumulation, DNA methylation and transcriptional gene silencing. EMBO J. 26, 16911701 (2007).
  115. Chandler, V. L. & Stam, M. Chromatin conversations: mechanisms and implications of paramutation. Nat. Rev. Genet. 5, 532544 (2004).
  116. Hollick, J. B. Paramutation: a trans-homolog interaction affecting heritable gene regulation. Curr. Opin. Plant Biol. 15, 536543 (2012).
  117. Luff, B., Pawlowski, L. & Bender, J. An inverted repeat triggers cytosine methylation of identical sequences in Arabidopsis. Mol. Cell 3, 505511 (1999).
  118. Melquist, S., Luff, B. & Bender, J. Arabidopsis PAI gene arrangements, cytosine methylation and expression. Genetics 153, 401413 (1999).
  119. Erhard, K. F. & Hollick, J. B. Paramutation: a process for acquiring trans-generational regulatory states. Curr. Opin. Plant Biol. 14, 210216 (2011).
  120. Belele, C. L. et al. Specific tandem repeats are sufficient for paramutation-induced trans-generational silencing. PLoS Genet. 9, e1003773 (2013).
  121. Du, J. et al. Dual binding of chromomethylase domains to H3K9me2-containing nucleosomes directs DNA methylation in plants. Cell 151, 167180 (2012).
  122. Law, J. A. et al. Polymerase IV occupancy at RNA-directed DNA methylation sites requires SHH1. Nature 498, 385389 (2013).
    Establishes the first direct link between H3K9me2 and Pol IV recruitment to methylated DNA via SHH1, as an essential step to initiate and/or maintain RdDM.
  123. Johnson, L. M. et al. SRA- and SET-domain-containing proteins link RNA polymerase V occupancy to DNA methylation. Nature 507, 124128 (2014).
  124. Bond, D. M. & Baulcombe, D. C. Epigenetic transitions leading to heritable, RNA-mediated de novo silencing in Arabidopsis thaliana. Proc. Natl Acad. Sci. USA 112, 917922 (2015).
  125. Washburn, J. D. & Birchler, J. A. Polyploids as a 'model system' for the study of heterosis. Plant Reprod. 27, 15 (2014).
  126. Chen, Z. J. Genomic and epigenetic insights into the molecular bases of heterosis. Nat. Rev. Genet. 14, 471482 (2013).
  127. Ng, D. W.-K., Lu, J. & Chen, Z. J. Big roles for small RNAs in polyploidy, hybrid vigor, and hybrid incompatibility. Curr. Opin. Plant Biol. 15, 154161 (2012).
  128. Groszmann, M., Greaves, I. K., Fujimoto, R., Peacock, W. J. & Dennis, E. S. The role of epigenetics in hybrid vigour. Trends Genet. 29, 684690 (2013).
  129. Ha, M. et al. Small RNAs serve as a genetic buffer against genomic shock in Arabidopsis interspecific hybrids and allopolyploids. Proc. Natl Acad. Sci. USA 106, 1783517840 (2009).
  130. Shivaprasad, P. V., Dunn, R. M., Santos, B. A., Bassett, A. & Baulcombe, D. C. Extraordinary transgressive phenotypes of hybrid tomato are influenced by epigenetics and small silencing RNAs. EMBO J. 31, 257266 (2011).
  131. Barber, W. T. et al. Repeat associated small RNAs vary among parents and following hybridization in maize. Proc. Natl Acad. Sci. USA 109, 1044410449 (2012).
  132. Shen, H. et al. Genome-wide analysis of DNA methylation and gene expression changes in two Arabidopsis ecotypes and their reciprocal hybrids. Plant Cell 24, 875892 (2012).
  133. Josefsson, C., Dilkes, B. & Comai, L. Parent-dependent loss of gene silencing during interspecies hybridization. Curr. Biol. 16, 13221328 (2006).
  134. Durand, S., Bouché, N., Perez Strand, E., Loudet, O. & Camilleri, C. Rapid establishment of genetic incompatibility through natural epigenetic variation. Curr. Biol. 22, 326331 (2012).
  135. Settles, A. M., Baron, A., Barkan, A. & Martienssen, R. A. Duplication and suppression of chloroplast protein translocation genes in maize. Genetics 157, 349360 (2001).
  136. Slotkin, R. K., Freeling, M. & Lisch, D. Heritable transposon silencing initiated by a naturally occurring transposon inverted duplication. Nat. Genet. 37, 641644 (2005).
    Provides the first example of a transposon-derived inverted repeat that is able to establish heritable epigenetic silencing of active transposons in the maize genome.
  137. Wei, W. et al. A role for small RNAs in DNA double-strand break repair. Cell 149, 101112 (2012).
    Demonstrates the involvement of small RNAs and AGO2 in DSB repair in A. thaliana.
  138. Lee, H.-C. et al. qiRNA is a new type of small interfering RNA induced by DNA damage. Nature 459, 274277 (2009).
  139. Kloc, A., Zaratiegui, M., Nora, E. & Martienssen, R. RNA interference guides histone modification during the S phase of chromosomal replication. Curr. Biol. 18, 490495 (2008).
  140. Zaratiegui, M. et al. RNAi promotes heterochromatic silencing through replication-coupled release of RNA Pol II. Nature 479, 135138 (2011).
  141. Oliver, C., Santos, J. L. & Pradillo, M. On the role of some ARGONAUTE proteins in meiosis and DNA repair in Arabidopsis thaliana. Front. Plant Sci. 5, 177 (2014).
  142. Michalik, K. M., Böttcher, R. & Förstemann, K. A small RNA response at DNA ends in Drosophila. Nucleic Acids Res. 40, 95969603 (2012).
  143. Francia, S. et al. Site-specific DICER and DROSHA RNA products control the DNA-damage response. Nature 488, 231235 (2012).
  144. Chiolo, I. et al. Double-strand breaks in heterochromatin move outside of a dynamic HP1a domain to complete recombinational repair. Cell 144, 732744 (2011).
  145. Gao, M. et al. Ago2 facilitates Rad51 recruitment and DNA double-strand break repair by homologous recombination. Cell Res. 24, 532541 (2014).
  146. Kapoor, M. et al. Genome-wide identification, organization and phylogenetic analysis of Dicer-like, Argonaute and RNA-dependent RNA polymerase gene families and their expression analysis during reproductive development and stress in rice. BMC Genomics 9, 451 (2008).
  147. Singh, M. et al. Production of viable gametes without meiosis in maize deficient for an ARGONAUTE protein. Plant Cell 23, 443458 (2011).
    Details a genetic screen in maize that uncovered ago104 mutants that mimic apomixis by forming viable unreduced gametes.
  148. Borges, F., Pereira, P. A., Slotkin, R. K., Martienssen, R. A. & Becker, J. D. MicroRNA activity in the Arabidopsis male germline. J. Exp. Bot. 62, 16111620 (2011).
  149. Olmedo-Monfil, V. et al. Control of female gamete formation by a small RNA pathway in Arabidopsis. Nature 464, 628632 (2010).
    Describes a non-cell-autonomous function of small RNAs and AGO9 during differentiation and specification of female gametes in A. thaliana.
  150. Komiya, R. et al. Rice germline-specific Argonaute MEL1 protein binds to phasiRNAs generated from more than 700 lincRNAs. Plant J. 78, 385397 (2014).
  151. Tucker, M. R. et al. Somatic small RNA pathways promote the mitotic events of megagametogenesis during female reproductive development in Arabidopsis. Development 139, 13991404 (2012).
  152. Nonomura, K. I. et al. A germ cell specific gene of the ARGONAUTE family is essential for the progression of premeiotic mitosis and meiosis during sporogenesis in rice. Plant Cell 19, 25832594 (2007).
  153. Fu, Q. & Wang, P. J. Mammalian piRNAs: biogenesis, function, and mysteries. Spermatogenesis 4, e27889 (2014).
  154. Poulsen, C., Vaucheret, H. & Brodersen, P. Lessons on RNA silencing mechanisms in plants from eukaryotic argonaute structures. Plant Cell 25, 2237 (2013).
  155. Mi, S. et al. Sorting of small RNAs into Arabidopsis Argonaute complexes is directed by the 5′ terminal nucleotide. Cell 133, 116127 (2008).
    Describes the first study showing that biased small-RNA sorting into AGOs in A. thaliana is regulated by the 5′-terminal nucleotides of small-RNA duplexes.
  156. Takeda, A., Iwasaki, S., Watanabe, T., Utsumi, M. & Watanabe, Y. The mechanism selecting the guide strand from small RNA duplexes is different among argonaute proteins. Plant Cell Physiol. 49, 493500 (2008).
  157. Frank, F., Hauver, J., Sonenberg, N. & Nagar, B. Arabidopsis Argonaute MID domains use their nucleotide specificity loop to sort small RNAs. EMBO J. 31, 35883595 (2012).
  158. Zhu, H. et al. Arabidopsis Argonaute10 specifically sequesters miR166/165 to regulate shoot apical meristem development. Cell 145, 242256 (2011).
    Demonstrates that the preference of AGO10 for miR165 and miR166 relies on the structure of the mature small-RNA duplex and is essential to promote shoot apical meristem development.
  159. Liu, Q. et al. The ARGONAUTE10 gene modulates shoot apical meristem maintenance and establishment of leaf polarity by repressing miR165/166 in Arabidopsis. Plant J. 58, 2740 (2009).
  160. Ji, L. et al. ARGONAUTE10 and ARGONAUTE1 regulate the termination of floral stem cells through two microRNAs in Arabidopsis. PLoS Genet. 7, e1001358 (2011).
  161. Montgomery, T. A. et al. Specificity of ARGONAUTE7–miR390 interaction and dual functionality in TAS3 trans-acting siRNA formation. Cell 133, 128141 (2008).
  162. Endo, Y., Iwakawa, H.-O. & Tomari, Y. Arabidopsis ARGONAUTE7 selects miR390 through multiple checkpoints during RISC assembly. EMBO Rep. 14, 652658 (2013).
    Reports the extensive characterization of structural determinants in the conserved miR390 mature duplex and their importance for preferential loading onto AGO7 and the formation of a functional RNA-induced silencing complex.
  163. Zhang, X. et al. Arabidopsis Argonaute 2 regulates innate immunity via miRNA393*-mediated silencing of a Golgi-localized SNARE gene, MEMB12. Mol. Cell 42, 356366 (2011).
  164. Zhang, X. et al. ARGONAUTE PIWI domain and microRNA duplex structure regulate small RNA sorting in Arabidopsis. Nat. Commun. 5, 5468 (2014).
    Provides further mechanistic insight and functional significance for sorting guide and passenger strands of miRNA duplexes into AGO1 and AGO2, respectively.
  165. Chitwood, D. H. et al. Pattern formation via small RNA mobility. Genes Dev. 23, 549554 (2009).
  166. Molnar, A. et al. Small silencing RNAs in plants are mobile and direct epigenetic modification in recipient cells. Science 328, 872875 (2010).
  167. Skopelitis, D. S., Husbands, A. Y. & Timmermans, M. C. P. Plant small RNAs as morphogens. Curr. Opin. Cell Biol. 24, 217224 (2012).
  168. Carlsbecker, A. et al. Cell signalling by microRNA165/6 directs gene dose-dependent root cell fate. Nature 465, 316321 (2010).
  169. Sarkies, P. & Miska, E. A. Small RNAs break out: the molecular cell biology of mobile small RNAs. Nat. Rev. Mol. Cell Biol. 15, 525535 (2014).
  170. Mosher, R. A. et al. Uniparental expression of PolIV-dependent siRNAs in developing endosperm of Arabidopsis. Nature 460, 283286 (2009).
  171. Calarco, J. P. et al. Reprogramming of DNA methylation in pollen guides epigenetic inheritance via small RNA. Cell 151, 194205 (2012).
  172. Ibarra, C. A. et al. Active DNA demethylation in plant companion cells reinforces transposon methylation in gametes. Science 337, 13601364 (2012).
  173. Vu, T. M. et al. RNA-directed DNA methylation regulates parental genomic imprinting at several loci in Arabidopsis. Development 140, 29532960 (2013).
  174. Borsani, O., Zhu, J., Verslues, P. E., Sunkar, R. & Zhu, J.-K. Endogenous siRNAs derived from a pair of natural cis-antisense transcripts regulate salt tolerance in Arabidopsis. Cell 123, 12791291 (2005).
  175. Katiyar-Agarwal, S., Gao, S., Vivian-Smith, A. & Jin, H. A novel class of bacteria-induced small RNAs in Arabidopsis. Genes Dev. 21, 31233134 (2007).
  176. Ron, M., Alandete Saez, M., Eshed Williams, L., Fletcher, J. C. & McCormick, S. Proper regulation of a sperm-specific cis-nat-siRNA is essential for double fertilization in Arabidopsis. Genes Dev. 24, 10101021 (2010).
  177. Felippes, F. F. & Weigel, D. Triggering the formation of tasiRNAs in Arabidopsis thaliana: the role of microRNA miR173. EMBO Rep. 10, 264270 (2009).
  178. McCue, A. D., Nuthikattu, S., Reeder, S. H. & Slotkin, R. K. Gene expression and stress response mediated by the epigenetic regulation of a transposable element small RNA. PLoS Genet. 8, e1002474 (2012).

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Affiliations

  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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