Cell-free reconstitution reveals the molecular mechanisms for the initiation of secondary siRNA biogenesis in plants

Abstract

Secondary 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.

Nuclear and cytoplasmic RNA exosomes and PELOTA1 prevent miRNA-induced secondary siRNA production in Arabidopsis

“…Although the evidence against RISC-generated cleavage fragments as triggers of siRNA production via RDR6 from this and previous work ( 11 , 42 , 78 , 84 , 85 ) is now compelling, aberrant RNA clearly can play a role in RDR-mediated siRNA amplification given the many reports of ectopic siRNA production in mutants in mRNA decay ( 33–41 ). How may these seemingly opposing pieces of evidence be reconciled?…”

“…Likewise, AGO7-miR390 is capable of triggering TAS3 secondary siRNAs from uncleavable miR390 sites in TAS3 precursor RNAs ( 78 ). In addition, recent recapitulation of AGO1-miR173- and AGO7-miR390-triggered tasiRNA generation in cell-free systems showed that RDR6 and AGO physically associate in RISC:target associations leading to tasiRNA production, thus verifying a central element of the RISC trigger model ( 79 , 80 ). In the following, we offer an interpretation of the results described here within the framework of the RISC-trigger model.…”

High‐pressure sprayed siRNAs influence the efficiency but not the profile of transitive silencing

“…Indeed, 22‐nt siRNAs and/or siRNAs exhibiting an asymmetric bulge at their duplex were supposed to change AGO1 conformation (Manavella et al, 2012). This new conformation could not only impair the RNase activity of AGO1, but could also recruit RDR6 to the 3ʹ of the target transcript, conceivably with the aid of mediators such as SDE3 and SGS3 (Garcia et al, 2012; Sakurai et al, 2021). Although 22‐nt miRNAs are demonstrated to cleave the target RNAs, our study shows 22‐nt siRNAs have evidently a different mode of action than 22‐nt miRNAs.…”

“…The resulting primary dsRNAs are processed into siRNAs, initiating target RNA silencing (Parent et al, 2015; Wassenegger and Krczal, 2006). In addition, it has been demonstrated that RDR6 is recruited to transcripts that are targeted by (primary) siRNAs for the generation of dsRNA and secondary siRNAs, which map upstream and downstream of the primary siRNA site, a process termed transitivity (Bleys et al, 2006a,b; Chen et al, 2010; Cuperus et al, 2010; Koscianska et al, 2005; Manavella et al, 2012; Mlotshwa et al, 2008; Moissiard et al, 2007; Montgomery et al, 2008a,b; Sakurai et al, 2021; Vaistij and Jones, 2009; Vaistij et al, 2002; Vermeersch et al, 2010, 2013).…”

“…Along with the intrinsic features of the target transcript, the sRNA trigger itself appears to affect the initiation and efficiency of transitive silencing. It has been suggested that sRNAs with a size of 22‐nt (Chen et al, 2010) or sRNAs containing an asymmetric bulge in their duplex (Manavella et al, 2012) are changing AGO1 conformation thereby recruiting RDR6 to their target, most likely with the aid of mediators such as SDE3 (Garcia et al, 2012), SDE5 and SGS3 (Sakurai et al, 2021). In line with this, DCL2, responsible for the generation of 22‐nt siRNAs, is required for transitivity initiation (Mlotshwa et al, 2008).…”