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Exploring new models of easiRNA biogenesis Alexis Sarazin & Olivier Voinnet


© 2014 Nature America, Inc. All rights reserved.

Although silent transposons in plants can be reactivated by stress or during development, their potential deleterious effects are prevented by transposon-derived epigenetically activated small interfering RNAs (easiRNAs). A new study shows how serendipitous interactions between reactivated transposons and endogenous microRNAs might initiate easiRNA biogenesis, establishing an unexpected link between these two classes of silencing small RNAs. Intertwined epigenetic processes protect plant genomes from the deleterious effects of transposable elements (TEs). In Arabidopsis thaliana, some TEs are silenced via cytosine methylation and chromatin compaction mediated by DNA METHYLTRANSFERASE-1 (MET1) and DECREASED DNA METHYLATION-1 (DDM1), but silencing can be reset developmentally or inactivated by stress. Therefore, backup mechanisms exist to contain epigenetically reactivated TEs, notably via conversion of their transcripts into double-stranded RNA (dsRNA) by RNA-dependent RNA polymerase 6 (RDR6). The Dicer-like RNase III enzymes, DCL4 and DCL2, process the dsRNA into populations of 21- and 22-nt easiRNAs that, upon loading into AGO1 and AGO2, are thought to guide post-transcriptional gene silencing of many reactivated TEs1–3 (Supplementary Fig. 1). How RDR6 initiates easiRNA biogenesis specifically on TE RNAs has remained mysterious, but new work reported by Robert Martienssen and colleagues in Nature 4 could provide part of the answer by implicating endogenous microRNAs (miRNAs) in this process. Exception to the rule Most plant miRNAs are produced by DCL1 from primary transcripts containing imperfect self-complementary fold-back regions. They regulate mRNAs usually bearing a single miRNA-complementary target site by promoting their endonucleolytic cleavage5. Cleaved RNA fragments are normally degraded, but, under some circumstances, RDR6 uses them as templates to initiate the production of trans-acting siRNAs (tasiRNAs) Alexis Sarazin and Olivier Voinnet are in the Department of Biology at the Swiss Federal Institute of Technology Zürich, Zürich, Switzerland. e-mail: [email protected]


via mechanisms resembling those of easiRNA biogenesis6. Notably, tasiRNAs enable control of a much wider range of related transcripts than individual miRNAs alone, a feature welltailored to the predicted role of easiRNAs. Creasey et al.4 thus hypothesized that miRNAs could similarly initiate RDR6 action on epigenetically reactivated TE transcripts, a situation artificially created in ddm1 mutant Arabidopsis. The global easiRNA levels found in ddm1 mutants were indeed lower in ddm1-dcl1 double mutants and, as expected, were nearly abolished in ddm1-rdr6 double mutants. Differential small RNA profiling in ddm1 versus ddm1-rdr6 mutant inflorescences identified many potential easiRNA-generating miRNAs, for which 3,662 possible targets were bioinformatically predicted among the 3,903 annotated Arabidopsis TE genes. This high initial estimate likely reflects the relaxed miRNA-pairing parameters used for target prediction and was indeed halved following parallel analysis of RNA ends (PARE) set to retrieve discrete miRNA cleavage products on a genome-wide scale. Further mapping of potential 21- and 22-nt easiRNAs, validated with a low-read threshold, finally showed that approximately one-third of all TE gene transcripts possibly undergo miRNA-mediated cleavage and simultaneously spawn easiRNAs in ddm1 mutant inflorescences. Although likely an overestimation, this figure concurs with previous findings that only 15 of ∼320 TE subfamilies contribute substantially to easiRNA production in ddm1 mutants yet occupy, alone, ∼25% of the total TE space2. However, it is clear from the authors’ data that only some sequences within this subset, or even within a given TE subfamily, are associated with easiRNAs. TEs themselves are unlikely to contribute substantially to this apparent specificity in targeting, as transposons with dissimilar evolutionary states or proliferation mechanisms spawn easiRNAs. Instead, atypical

features of easiRNA-generating miRNAs, evident in the authors’ data set, are likely involved. RNA quality control (RQC) mediated by 5′–3′ and 3′–5′ exonucleases normally presents a robust barrier to the pervasive access of RDR6 to miRNA-cleaved fragments7; however, RQC operates poorly on RNA substrates lacking both a 5′ cap and a 3′ polyA tail. Such substrates can be generated by multiple, discrete miRNA cuts known to stimulate RDR6-dependent tasiRNA production6,7. Single-cut miRNAs can also trigger RDR6 activity if they are 22 nt instead of 20 or 21 nt in length (the cognate size range of most miRNAs) or if their precursors display specific structural features8,9 (Supplementary Fig. 1). Simultaneous multiple cuts by miRNAs are infrequent, and only a fraction of single-cut miRNAs would possess the necessary attributes to attract RDR6 on matching, reactivated TE transcripts. Additionally, given that plant miRNA action is poorly tolerant to imperfect base-pairing5, easi­ RNA production triggered by miRNAs is probably more an exception than the rule, and this feature likely contributes to shaping the easiRNA landscape observed in ddm1 mutant plants. Serendipity versus adaptive coevolution Experiments conducted in ddm1 mutants draw an ideal picture of easiRNA production that poorly reflects the spatiotemporal confinement of stresses or developmental programs that would normally reactivate TEs in wild-type plants (Fig. 1a). For instance, a transient deficit in DDM1 naturally promotes easiRNA production in pollen vegetative nuclei, where only a fraction of the ∼50 predicted easiRNAgenerating miRNAs are expressed1,10. Moreover, many prevalent easiRNA-generating miRNAs are highly conserved across plant species, with their basic roles in the acquisition or maintenance of cell identity confining their accumulation within single tissues or even single cells in a manner inconsistent with their mobility11,12 (Fig. 1a). Thus, a model for easiRNA production

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Chromatin compaction and DNA methylation


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© 2014 Nature America, Inc. All rights reserved.


Figure 1 Models for easiRNA biogenesis triggered by miRNAs. (a) Serendipity-based model involving an ancient miRNA confined within a specific domain of the shoot apical meristem (SAM). In case 1, a TE locally reactivated by stress in wild-type (WT) plants is unable to engage easiRNA biogenesis because its expression pattern is outside the SAM, unlike in case 2, which depicts a distinct stressinduced TE. In ddm1 mutants, depicted in case 3, many TEs would be reactivated in a stressindependent manner, providing many more opportunities to initiate easiRNA biogenesis. (b) In a coevolution model, an inversion-duplication event contemporary to the TE genomic proliferation phase spawns a young TE-derived miRNA locus. Ensuing miRNA-TE coevolution would adapt the miRNA for optimal easiRNA production, for instance, through the selection of rare 22-nt isoforms. All contenders would eventually undergo stress-reversible epigenetic silencing via chromatin compaction by DDM1 and DNA methylation (red circles) by MET1.

based on serendipitous interactions between ancient miRNAs and reactivated TE transcripts is not easily accommodated in wild-type plants (Fig. 1a), particularly if rare 22-nt miRNA isoforms or double-cuts are conditional for RDR6 recruitment (Supplementary Fig. 1). The model also holds limited scope in bringing the targeted TEs under robust miRNA control through adaptive evolution. A more conceivable model, in our opinion, posits that easiRNA-generating miRNAs are spawned during the initial genomic expansion phase of TEs, which would occasionally be accompanied by inversion-duplication events (Fig. 1b). As proposed for the birth of conventional plant miRNA genes13, invertedduplicated TE loci could provide raw material for the evolution and selection of TE-derived miRNAs with optimal easiRNA-generating properties. This scheme would potentially bring the TE-derived miRNA locus and the TE target loci under the same epigenetic control and confine them within the same developmental and spatial niches. As a result, easiRNA production and epigenetic reactivation of target TEs would coincide precisely (Fig. 1b). Consistent with this coevolution model, Creasey et al. identified several epigenetically reactivated miRNAs (eamiRNAs) in ddm1 mutants, of which at

least one was derived from an ATHILA4 retro­ element. Related TE-based scenarios have already been proposed to explain the possible origins of miRNA genes5, raising the possibility that, just like siRNAs, miRNAs might have primarily evolved for defensive rather than regulatory purposes. TE-derived (ea)miRNAs would likely undergo positive selection because they benefit the host but also, possibly, the founder TEs themselves. Indeed, the authors observed that many easiRNA-generating TE loci produced 24-nt siRNAs instead of 21- or 22-nt siRNAs and gained DNA methylation in ddm1-rdr6 double mutants. siRNAs of 24 nt in length are synthesized by DCL3 from dsRNA produced by RDR2 (ref. 14), which competes with RDR6 for substrates15, and guide cytosine methylation, potentially resulting in heritable transcriptional gene silencing of their loci of origin. As pointed out by Creasey et al., TEs might tolerate transient post-transcriptional gene silencing by easiRNAs as a compromise to avoid long-term transcriptional gene silencing by methylation. The existence of this pathway for transcriptional gene silencing also suggests that studies of rdr2-rdr6 double mutants or rdr1-rdr2-rdr6 triple mutants might clarify the still poorly understood function of the

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easiRNAs accumulating naturally in pollen vegetative nuclei1. Conclusions and future prospects miRNA-triggered easiRNAs are a thoughtprovoking concept with important implications in transposon biology, evolution and epigenetic reprogramming. Nonetheless, the molecular tenets of this concept entail an unusual combination of circumstances and/ or a degree of coevolution that is unlikely to apply commonly to epigenetically reactivated TEs. Indeed, only a fraction of easiRNA­associated TEs showed evidence of cleavage by PARE and vice versa4; thus, additional mechanisms surely underpin easiRNA biogenesis. One possible lead to identifying these alternative mechanisms is through the reactivation of evolutionarily young TEs with low copy numbers and, ideally, narrow expression patterns. The Arabidopsis singlecopy retroelement éVADé (EVD) fulfils these criteria, and its epigenetic reactivation in met1 or ddm1 mutants triggers a sharply defined cohort of easiRNAs3 whose accumulation remains unaltered in ddm1-dcl1 double mutants, as determined from inspection of the authors’ data. Thus, a TE-intrinsic and miRNA-independent mechanism must underlie easiRNA production in this and probably many other cases. Elucidating this mechanism could solve the long-standing question of how de novo invading TEs with no sequence homology to the genome are primarily detected by their hosts. Note: Any Supplementary Information and Source Data files are available in the online version of the paper (doi:10.1038/ng.2993). COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. 1. Slotkin, R.K. et al. Cell 136, 461–472 (2009). 2. Nuthikattu, S. et al. Plant Physiol. 162, 116–131 (2013). 3. Marí-Ordóñez, A. et al. Nat. Genet. 45, 1029–1039 (2013). 4. Creasey, K.M. et al. Nature 508, 411–415 (2014). 5. Voinnet, O. Cell 136, 669–687 (2009). 6. Fei, Q., Xia, R. & Meyers, B.C. Plant Cell 25, 2400–2415 (2013). 7. Voinnet, O. Trends Plant Sci. 13, 317–328 (2008). 8. Chen, H.-M. et al. Proc. Natl. Acad. Sci. USA 107, 15269–15274 (2010). 9. Manavella, P.A., Koenig, D. & Weigel, D. Proc. Natl. Acad. Sci. USA 109, 2461–2466 (2012). 10. Borges, F., Pereira, P.A., Slotkin, R.K., Martienssen, R.A. & Becker, J.D. J. Exp. Bot. 62, 1611–1620 (2011). 11. Parizotto, E.A., Dunoyer, P., Rahm, N., Himber, C. & Voinnet, O. Genes Dev. 18, 2237–2242 (2004). 12. Válóczi, A., Várallyay, E., Kauppinen, S., Burgyán, J. & Havelda, Z. Plant J. 47, 140–151 (2006). 13. Allen, E. et al. Nat. Genet. 36, 1282–1290 (2004). 14. Kim, M.Y. & Zilberman, D. Trends Plant Sci. 19, 320–326 (2014). 15. Jauvion, V., Rivard, M., Bouteiller, N., Elmayan, T. & Vaucheret, H. PLoS ONE 7, e29785 (2012).


Exploring new models of easiRNA biogenesis.

Although silent transposons in plants can be reactivated by stress or during development, their potential deleterious effects are prevented by transpo...
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