Classification and Comparison of Small RNAs from Plants
RDR, DCL, and AGO proteins are three enzyme families cen- tral to plant small-RNA biogenesis and function. RDRs synthe- size second-strand RNA using an RNA template, resulting in the production of dsRNA. DCL endonucleases process helical RNA precursors (either dsRNA or the helical regions of stem- loop single-strandedRNAs) to release short double-stranded du- plexes, 20–24 nt long, with 2-nt 3' overhangs. AGOs then engage these duplexes, retaining only one of the two possible strands and discarding the other.AGO-loaded smallRNAs serve as specificity determinants to select RNA targets based on small-RNA/target complementarity. Successful target identification is followed by repressive activities orchestrated by the associated AGOprotein.
Hierarchical classification system for endogenous plant small RNAs.
Thick black lines indicate hierarchical relationships. Abbreviations: dsRNA, double-stranded RNA; hpRNA, hairpin RNA; miRNA, microRNA; NAT-siRNA, natural antisense transcript small interfering RNA; siRNA, small interfering RNA.
Patterns of functional microRNA (miRNA)/target complementarity in plants and animals
Note that a few additional marginal or atypical patterns of complementarity (10) that function in animals are not shown; none of these are known to function in plants.
Model for the biogenesis and function of heterochromatic small interfering RNAs (siRNAs) in plants.
Red dots show repressive chromatin marks such as 5-methyl cytosine and histone H3K9 methylation. Three outstanding questions are indicated with red stars. Adapted from Reference 113.
Cascades of secondary small interfering RNAs (siRNAs) can coordinately regulate a gene family
(a) Detailed schematic of secondary siRNA production.(b) Schematic cascade of secondary siRNA synthesis and targeting. An initial small RNA (magenta) targets a subset of a gene family, producing secondary siRNAs (small gray bars). Some of these are trans-acting siRNAs that can downregulate additional members of the gene family based on sequence complementarity. The process depicted in panel a occurs at each arrowhead in panel b. Additional abbreviations: dsRNA, double-stranded RNA; miRNA, microRNA.
The Diversity, Biogenesis, and Activities of Endogenous Silencing Small RNAs in Arabidopsis
Arabidopsis core silencing proteins
(a) Generic functions of DICER-LIKE (DCL), RNA-dependent RNA polymerase (RDR), HUA ENHANCER 1 (HEN1), and ARGONAUTE (AGO) proteins in small-RNA (sRNA) pathways. (b) Domain organization showing the conserved regions present in each class of core proteins. (c) Phylogenetic classification of Arabidopsis DCL, RDR, and AGO proteins. Protein sequences were aligned using MultAlin ([http://multalin.toulouse.inra.fr
competition and feedback regulation of silencing core proteins
(a) The microRNA (miRNA) pathway performs negative-feedback regulation of DICER-LIKE 1 (DCL1). (b) DCL proteins may compete for binding any given long double-stranded-RNA (dsRNA) substrate, generating small RNAs (sRNAs) of various lengths and 5′-end nucleotide identities (depicted here as w, x,y, and z letters), ultimately influencing their loading into specific ARGONAUTE (AGO) proteins. (c) AGO1 homeostatic regulation by miR168 may occur via AGO1-dependent slicing, AGO10-dependent translational repression, or increased miR168 accumulation in response to elevated AGO1 levels. (d) AGO10 and AGO1 compete for miR165/166 binding during shoot apical meristem development. Additional abbreviations: 7mG, 7-methylguanylate cap; An, polyadenine tail; HD-ZIP III, class-III homeodomain-leucine zipper; nt, nucleotide.
Posttranscriptional gene silencing–associated small-RNA (sRNA) pathways
(a) The Arabidopsis microRNA (miRNA) pathway. (b) Various types of miRNA-processing mechanisms. (c) The inverted repeat (IR)–derived small-interfering-RNA (siRNA) pathway. (d) The natural-antisense-transcript-siRNA (nat-siRNA) pathway. (e) Thetrans-acting-siRNA (tasiRNA) pathway. Additional abbreviations: 7mG, 7-methylguanylate cap; AGO, ARGONAUTE; An, polyadenine tail; CBP, CAP-BINDING PROTEINS; CPL1, C-TERMINAL DOMAIN PHOSPHATASE-LIKE 1; DCL, DICER-LIKE; DDL, DAWDLE; DRB4, double-stranded-RNA-binding protein 4; HEN1, HUA ENHANCER 1; HESO1, HEN1 SUPPRESSOR 1; HSP90, HEAT-SHOCK PROTEIN 90; HST, HASTY; HYL1, HYPONASTIC LEAVES 1; MOS2, MODIFIER OF SNC1 2; nt, nucleotide; Pol II, RNA polymerase II; pre-miRNA, precursor microRNA; pri-miRNA, primary microRNA; RDR6, RNA-dependent RNA polymerase 6; SDN1, SMALL RNA DEGRADING NUCLEASE 1; SE, SERRATE; SGS3, SUPPRESSOR OF GENE SILENCING 3; SIC, SICKLE; SQN, SQUINT; STA1, STABILIZED 1; P, phosphate; TGH, TOUGH.
Transcriptional gene silencing– and DNA-associated small-RNA (sRNA) pathways
(a) The repeat-associated small-interfering-RNA (rasiRNA) pathway. (b) The long-microRNA (lmiRNA) pathway. (c) The double-strand-break-induced-RNA (diRNA) pathway. Additional abbreviations: AGO, ARGONAUTE; CLSY1, CLASSY 1; DCL, DICER-LIKE; DMS3, DEFECTIVE IN MERISTEM SILENCING 3; DRD1, DEFECTIVE IN RNA-DIRECTED DNA METHYLATION 1; DRM, DOMAINS REARRANGED METHYLTRANSFERASE; DSB, double-strand break; HEN1, HUA ENHANCER 1; HSP90, HEAT-SHOCK PROTEIN 90; HST, HASTY; nt, nucleotide; Pol, RNA polymerase; RDR, RNA-dependent RNA polymerase; SHH1, SAWADEE HOMEODOMAIN HOMOLOG 1.
Subcellular localization and interaction of small-RNA pathway proteins
| Subcellular localizationc | ||||
|---|---|---|---|---|
| Proteina | Methodb | Cytosol | Nucleus | Colocalizationa |
| Core proteins | ||||
| DCL1 | FP | — | NO, NP bodies | HYL1, SE, CPL1, NOT2b |
| BiFC | — | NP bodies | HYL1, SE, TGH | |
| IF | —* | NP bodies (periNO)* | DCL3, DCL4, HEN1 | |
| DCL2 | FP | — | NP | — |
| IF | —* | NP bodies (periNO)* | — | |
| DCL3 | FP | — | NP | — |
| IF | — | NO bodies, NP bodies (periNO)* | AGO4-RDR2-NRPE1-DCLs-HEN1* | |
| DCL4 | FP | — | NP, NO | — |
| IF | —* | NP*, periNO | SGS3, AGO7 | |
| RDR2 | FP | — | NO bodies, NP bodies (periNO)* | DCL3, AGO4, NRPE1* |
| RDR6 | FP | Cyt bodies | — | SGS3, AGO7 |
| BiFC | Cyt bodies | — | SGS3 | |
| IF | Cyt bodies | NP | SGS3* | |
| HEN1 | FP | Cyt | NP, NP bodies | — |
| IF | —* | PeriNO* | DCL1, DCL3, DCL4* | |
| AGO1 | FP | Cyt bodies, ER mb | NP, NP bodies | HYL1, AGO1 |
| BiFC | — | NP bodies | HYL1 | |
| IF | —* | PeriNO* | — | |
| AGO4 | FP | — | NP | — |
| IF | —* | NO bodies, NP bodies (periNO)* | RDR2, DCL3, NRPE1* | |
| AGO5 | FP | Cyt | — | — |
| AGO7 | FP | Cyt bodies | — | RDR6, SGS3, DCL4* |
| IF | Cyt bodies | PeriNO* | RDR6, SGS3, DCL4* | |
| miRNA pathway | ||||
| HYL1 | FP | — | NP bodies, NP (less extension) | DCL1, SE, SIC, CPL1, MOS2, DRB4 |
| IF | — | NP bodies | DCL1, SE, AGO1, CPL1, SIC, TGH | |
| SE | FP | Cyt (less extension) | NP, NP bodies | HYL1, CPL1, RACK1 |
| BiFC | — | NP bodies | DCL1, HYL1, TGH, CPL1, NOT2b, SE | |
| CBP20 | BiFC | — | NP bodies | CBP80, SE, NOT2b |
| CBP80 | BiFC | — | NP bodies | CBP20, NOT2b |
| TGH | BiFC | — | NP bodies | DCL1, HYL1, SE |
| SIC | FP | — | NP bodies | HYL1 |
| IF | — | NP bodies | HYL1 | |
| CPL1 | FP | — | NP, NP bodies | HYL1, SE, DCL1 |
| BiFC | — | NP, NP bodies | HYL1, SE | |
| NOT2b | FP | — | NP | DCL1 |
| BiFC | — | NP bodies | SE, CBP20, CBP80 | |
| RACK1 | FP | — | NP | SE, AGO1 |
| BiFC | — | NP bodies | SE | |
| AMP1 | FP | ER mb | — | — |
| MOS2 | FP | — | NP | HYL1 |
| tasiRNA pathway | ||||
| SGS3 | FP | Cyt bodies | — | RDR6, AGO7 |
| BiFC | Cyt bodies | — | RDR6 | |
| IF | —* | NP, periNO | RDR6, SGS3, DCL4* | |
| DRB4 | FP | — | NP, NO | HYL1 |
| rasiRNA pathway | ||||
| DRM2 | IF | —* | NP* | — |
| Pol IV (NRPD1) | IF | —* | NP bodies* | AGO4, NRPD2, NRPE1* |
| Pol IV (NRPD2) | IF | —* | NP bodies* | AGO4, NRPD1, NRPE1* |
| Pol V (NRPE1) | IF | —* | NP, NO bodies* | AGO4, NRPD2 |
| DRD1 | IF | —* | NP* | — |
AGO, ARGONAUTE; AMP1, ALTERED MERISTEM PROGRAM 1; CBP, CAP-BINDING PROTEINS; CPL1, C-TERMINAL DOMAIN PHOSPHATASE-LIKE 1; DCL, DICER-LIKE; DRB4, double-stranded-RNA-binding protein 4; DRD1, DEFECTIVE IN RNA-DIRECTED DNA METHYLATION 1; DRM2, DOMAINS REARRANGED METHYLTRANSFERASE 1; HEN1, HUA ENHANCER 1; HYL1, HYPONASTIC LEAVES 1; miRNA, microRNA; MOS2, MODIFIER OF SNC1 2; NRPD1 Pol IV subunit NUCLEAR RNA POLYMERASE D1; NRPD2 Pol IV subunit NUCLEAR RNA POLYMERASE D2; NRPE1, Pol V subunit NUCLEAR RNA POLYMERASE E1; Pol, RNA polymerase; RACK1, RECEPTOR FOR ACTIVATED C KINASE 1; rasiRNA, repeat-associated small interfering RNA; RDR, RNA-dependent RNA polymerase; SE, SERRATE; SGS3, SUPPRESSOR OF GENE SILENCING 3; SIC, SICKLE; tasiRNA, trans-acting small interfering RNA; TGH, TOUGH.
BiFC, bimolecular fluorescence complementation; FP, fusion protein; IF, immunofluorescence.
Cyt, cytoplasm; ER mb, endoplasmic reticulum membrane; NO, nucleolus; NP, nucleoplasm; *, only isolated nuclei experiments were performed.
EN, endogenous expression; OE, overexpression; *, only isolated nuclei experiments were performed.
Ct, C terminus; Nt, N terminus; *, only isolated nuclei experiments were performed.
CFP, cyan fluorescent protein; GFP, green fluorescent protein; HA, human influenza hemagglutin; RFP, red fluorescent protein; YFP, yellow fluorescent protein; *, only isolated nuclei experiments were performed.
S, stable expression; T, transient expression.
