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Cell-free reconstitution reveals the molecular mechanisms for the initiation of secondary siRNA biogenesis in plants
Thursday, 2021/08/05 | 07:40:23
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Yuriki Sakurai, Kyungmin Baeg, Andy Y. W. Lam, Keisuke Shoji, Yukihide Tomari, and Hiro-oki Iwakawa PNAS August 3, 2021 118 (31) e2102889118 SignificanceDouble-stranded RNA (dsRNA) synthesis by RNA-dependent RNA polymerase (RDR) is a critical step in secondary small interfering RNA (siRNA) biogenesis. However, how RDR specifically converts the targets of primary small RNAs into dsRNA intermediates remains unclear. Here, we developed an in vitro system that recapitulates the production of secondary siRNAs that are physiologically important in plants. Leveraging this system, we showed that a combination of four plant factors promotes physical recruitment of RDR6 to the target RNA. Moreover, we found that dsRNA synthesis by RDR6 is enhanced by the removal of the poly(A) tail, which is achieved by cleavage at another small RNA-binding site bearing appropriate mismatches. Our data elucidate the molecular events necessary for secondary siRNA biogenesis in plants. AbstractSecondary small interfering RNA (siRNA) production, triggered by primary small RNA targeting, is critical for proper development and antiviral defense in many organisms. RNA-dependent RNA polymerase (RDR) is a key factor in this pathway. However, how RDR specifically converts the targets of primary small RNAs into double-stranded RNA (dsRNA) intermediates remains unclear. Here, we develop an in vitro system that allows for dissection of the molecular mechanisms underlying the production of trans-acting siRNAs, a class of plant secondary siRNAs that play roles in organ development and stress responses. We find that a combination of the dsRNA-binding protein, SUPPRESSOR OF GENE SILENCING3; the putative nuclear RNA export factor, SILENCING DEFECTIVE5, primary small RNA, and Argonaute is required for physical recruitment of RDR6 to target RNAs. dsRNA synthesis by RDR6 is greatly enhanced by the removal of the poly(A) tail, which can be achieved by the cleavage at a second small RNA-binding site bearing appropriate mismatches. Importantly, when the complementarity of the base pairing at the second target site is too strong, the small RNA–Argonaute complex remains at the cleavage site, thereby blocking the initiation of dsRNA synthesis by RDR6. Our data highlight the light and dark sides of double small RNA targeting in the secondary siRNA biogenesis.
See: https://www.pnas.org/content/118/31/e2102889118
Figure 1: Recapitulation of the TAS3 tasiRNA biogenesis pathway in vitro. (A) Schematic outlining current understanding of the TAS3 tasiRNA biogenesis pathway. AGO7-miR390 complexes target two binding sites in the TAS3 mRNA. The base pairings between miR390 (shown in red) and the 5′ and 3′ binding sites (shown in orange) possess the evolutionarily conserved mismatches (highlighted in light blue) at the central and the 3′ end region, respectively. Following AGO7-RISC–mediated cleavage of the 3′ proximal site, RDR6 converts the 5′ cleaved fragment into dsRNA. The resulting long dsRNA is then processed into 21-nt siRNAs by DCL4. 5′D7 (+) tasiRNA, which is the seventh tasiRNA in register with the cleavage site, is located in the sense strand of the long dsRNA. (B) Flowchart of TAS3 tasiRNA biogenesis assay. After in vitro translation of mRNAs encoding tasiRNA factors in BY-2 lysate, miR390 duplex was added to form AGO7-RISC. The TAS3 mRNA was then added to trigger tasiRNA biogenesis. (C) In vitro recapitulation of TAS3 tasiRNA biogenesis. tasiRNA biogenesis was performed as outlined in B. TAS3 sense RNA and 5′D7 (+) tasiRNA were detected by northern blotting. U6 spliceosomal RNA was used as a loading control. See also SI Appendix, Fig. S1B. (D) Quantification of 5′D7 (+) tasiRNAs in C. The band intensity of 5′D7 (+) tasiRNA was normalized to the value of that in the reaction mixture with all tasiRNA factors (All). The graphs show the mean ± SD from five technically independent experiments. Bonferroni-corrected P values from two-sided paired t test are indicated (*P = 4.92 × 10−4, **P = 3.66 × 10−3, ***P = 1.45 × 10−3). (E) Distribution of 21-nt siRNAs relative to TAS3a mRNA produced in recapitulated reaction mixtures (AGO7 only, miR390 only, and AGO7 + miR390), in Col-0, and in sgs3-11 seedlings (top to bottom). The traces indicate the position of the 5′ end for sense siRNAs and antisense siRNAs. The 5′ end of miR390 binding sites and 3′ cleavage sites are indicated by orange lines. In the presence of AGO7-miR390-RISC, siRNAs efficiently mapped between the 5′ and 3′ miR390 binding sites in the TAS3 mRNA. siRNAs mapped to the TAS3 loci in Col-0 but not in the sgs3-11 mutant. See also SI Appendix, Fig. S1E. |
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