DICER-LIKE1 processed trans-acting siRNAs mediate DNA methylation

Abstract

Small non-coding RNAs (sRNAs) emerge as exquisite molecules that are guided for transcriptional and posttranscriptional gene regulation in eukaryotes. As one class of most important sRNAs in plants, trans-acting small interfering RNAs (ta-siRNAs) initiate from microRNA (miRNA) – mediated cleavage of TAS gene transcripts and subsequently are stabilized by SUPPRESSOR OF GENE SILENCING3 (SGS3) and converted to double–stranded RNA (dsRNA) by the actions of RNA-DEPENDENT RNA POLYMERASE6 (RDR6). Generally, these dsRNAs are processed by DICER-LIKE4 (DCL4) and recruited into ARGONAUTE 1 (AGO1) complexes to posttranscriptionally regulate target genes by mRNA cleavage in trans. In a recent study, we discovered a non-canonical ta-siRNAs pathway: Starting from the miRNA-guided cleavage site, the dsRNAs are processed by DCL1 into 21-nt siRNAs, which associate with AGO4/6 complexes to direct DNA methylation in cis. Together with previous results that miRNAs can be produced by DCL3, loaded into AGO4 and trigger epigenetically regulation of target genes, these findings indicate much complex biogenesis, effector and action pathways exist in plant sRNAs kingdom.

Keywords: :

• ta-siRNA
• DCL
• AGO
• DNA methylation
• non-canonical pathway

This article refers to:

Plant occupy small silencing RNAs, containing microRNAs (miRNAs) and small interfering RNAs (siRNAs), to exquisitely regulate target genes in a wide range of development processes and responses to abiotic and biological stresses. So far, plant endogenous siRNAs have been reported as three subcategories: heterochromatic siRNAs (hc-siRNAs), natural antisense transcript-derived siRNAs (nat-siRNAs) and trans-acting-siRNAs (ta-siRNAs).¹ Typically, miRNAs negatively regulate targets by mRNA cleavage and translation inhibition in trans; hc-siRNAs silence transponson and repeat elements though DNA methylation and histone modification in cis; nat-siRNAs are induced when plants grow in abnormal conditions and cause the silencing of antisense transcripts via disruption of mRNA stability in cis; the silencing fashions of ta-siRNAs like miRNAs, which usually modulate targets as slicers in trans.²

DCLs and AGOs are recognized as key players in sRNA biogenesis and effector pathways. Plants harbor four classes of DCL proteins and three clades of AGO proteins. A decade ago, it is known that DCL1 is responsible for miRNAs formation from hairpin structures precursors and AGO1 is the effector complexes essential for miRNA-mediated targets mRNA cleavage, since dcl1 and ago1 mutants displayed pleiotropic phenotypes as loss of miRNA activities.^3,4 Afterwards, people found the biogenesis of some newly evolved miRNAs in Arabidosis requires DCL4 and miRNA can function as translation repressors through AGO1, which provided insights for wide miRNA biogensis and action pathways.^5,6 Recently, our studies in rice revealed that a class of 24-nt long miRNAs are produced by DCL3 and sorted into AGO4 clades proteins to direct DNA methylation for targets epigenetic regulation, which greatly expands the knowledge of complex miRNA production and effect pathways.⁷

As a plant-specific siRNAs, ta-siRNAs appear to be hallmark of secondary siRNAs, which initiate from miRNA-cleavaged TAS1–4 gene transcripts.⁸ These cleavage products are then stabilized by SGS3, copied into dsRNAs by RDR6, sequentially processed by DCL4 and loaded into AGO1, resulting in target mRNA degradation.^9,10

In recent years, most publications regarding ta-siRNAs biogenesis centered on discovery of ta-siRNA formation reasons and a plenty of mechanisms have been raised, such as “two-hit” models, “22-nt miRNAs as specific ta-siRNA initiators,” “asymmertrical bulged bases between miRNAs and miRNA duplex as sufficient structures for ta-siRNA transitivity,” however, SGS3-RDR6-DCL4, which was found nearly a decade years ago, are still considered as the sole pathway for ta-siRNA biogenesis.^11-13 Moreover, although genome-wide DNA methylome sequencing studies detected cytosine methylation occurred in TAS gene loci, the reasons and mechanisms are largely unknown.¹⁴

In recent work, though bisulfite sequencing analysis, we detected high cytosine DNA methylation confined at regions close to the miRNA cleavage site of TAS genes, suggesting that ta-siRNAs may have alternative actions that trigger DNA methylation for transcriptional regulation. As expected, SGS3 and RDR6, the key players for ta-siRNA formation, are found indispensable for TAS-derived siRNA directed DNA methylation. However, another important component for ta-siRNA generation, DCL4, is not required during this process.¹⁵ This unexpected result provides an opportunity for us to test whether other DCL proteins involve in ta-siRNA biogenesis. Although it has been reported that DCL2, DCL3 and DCL4 can function redundantly in ta-siRNA production,¹⁶ our further studies showed DNA methylation at TAS3 locus still persists in dcl2, dcl3, dcl4 double and triple mutants. Since DCL1 is also required for miRNA production, previous research cannot define the dicing role of DCL1 to TAS transcript is direct or indirect. Hence, we first determined the exact role of DCL1 in the process of ta-siRNA production: Besides miRNA formation, DCL1 can directly process TAS transcripts to produce TAS siRNAs for mediation of DNA methylation. These findings indicate both DCL1 and DCL4 can process TAS gene dsRNA transcripts.¹⁵

Another important question is that ta-siRNAs interact with which AGO proteins to trigger DNA methylation. High-throughput sequencing results have revealed that most of ta-siRNAs from TAS loci were sorted into AGO1 and AGO2,¹⁷ however, cytosine methylation triggered by ta-siRNAs are not compromised in ago1 and ago2 mutants.¹⁵ Although only a small number of TAS siRNA are loaded into AGO4 and AGO6, our genetic analyses found that the interaction of AGO4 clade proteins and ta-siRNAs are sufficient to trigger epigenetic regulation. These results suggest besides of the 5′ terminal nucleotide and their biogenesis pathway, sRNAs loading into AGOs is also determined by their functions to some extent.^7,15,17 There may be a signal that can connect with sRNAs subsequent loading and ongoing functions.

In plants, silencing at endogenous transposons and repeats loci is usually guided by homologous 24-nt hc-siRNAs via RNA-directed DNA methylation (RdDM) pathway.¹⁸ However, 21-nt ta-siRNA guided DNA methylation are not dependent on upstream RdDM pathway players (Pol IV, DCL3, RDR2), but strictly relied on downstream players (AGO4, Pol V, and so on), suggesting that each type of siRNA triggered DNA methylation utilize the same effector counterparts in RdDM pathway.¹⁵

Although we unveiled the exact biogenesis and effector players for ta-siRNA mediated DNA methylation pathway, however, the precise biological role of this methylation remains obscure because no change of TAS gene expression took place when the methylation level was dramatically reduced.¹⁵ Moreover, we detected the DNA methylation level of TAS3 ta-siRNA targets (ARF2 and ARF3) loci, but neither CHG nor CHH methylation were found around the target sites (Fig. 1A and 1B). Therefore, other investigations in the coming years are essential to explore precise modulation roles of ta-siRNA guided DNA methylation.

Figure 1.DNA methylation level at TAS3 ta-siRNA target gene loci. A, Analysis of DNA methylation at ARF2 gene locus by bisulfite sequencing in Col-0 plants .B, ARF3 locus DNA methylation. Sequencing data were analyzed by using Kismeth software¹⁹ (http://katahdin.cshl.edu/homepage/kismeth/revpage.pl). The colored lines above the x axis show the percent methylation at individual cytosine sites. The positions corresponding to the tasiR-ARF sites are designated by grey bars (yellow bars in the online version).

In conclusion, our here publication of DCL1-AGO4-DNA methylation for ta-siRNAs and previous work on DCL3-AGO4-DNA methylation for miRNAs indicate the existence of much complex non-canonical small RNA biogenesis and action pathways in plants (Fig. 2).

Figure 2. Schematic model for complex biogenesis and action pathway of miRNAs and ta-siRNAs. Two distinct pathways for miRNAs and ta-siRNAs that operate at mRNA and chromatin levels are shown. In the left box, 21-nt miRNAs are typically produced by DCL1 and loaded into AGO1 to direct target genes mRNA cleavage or protein translation inhibition. Alternatively, a class of 24-nt miRNAs are generated by DCL3 and recruited into AGO4 to guide DNA methylation transcriptional regulation. In the right box, TAS gene transcripts are initiated by miRNA-AGO1/7 interacting cleavage complexes and then stabilized and converted into dsRNA by SGS3 and RDR6. Most of dsRNAs are processed by DCL4 to generate 21-nt ta-siRNAs associating with AGO1 for targets posttranscriptional modulation. Meanwhile, some of dsRNAs are processed by DCL1 to produce siRNAs interacting with AGO4 clade proteins and Pol V-scaffold RNA complexes for trigging epigenetic regulation though DNA methylation.

Acknowledgments

This work was supported by the the National “863” program of Ministry of Science and Technology of China (#2012AA10A308), the National Transgenic Key Project (#2011ZX08009–001), and a grant from the Beijing Municipal Government.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.