RRC ID 68947
Author Sakurai Y, Baeg K, Lam AYW, Shoji K, Tomari Y, Iwakawa HO.
Title Cell-free reconstitution reveals the molecular mechanisms for the initiation of secondary siRNA biogenesis in plants.
Journal Proc Natl Acad Sci U S A
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.
Volume 118(31)
Published 2021-8-3
DOI 10.1073/pnas.2102889118
PII 2102889118
PMID 34330830
PMC PMC8346886
MeSH Cell Line Cell-Free System Gene Expression Regulation, Plant / physiology* Plant Proteins / genetics Plant Proteins / metabolism* RNA Interference RNA, Small Interfering* Tobacco / cytology*
IF 9.412
Arabidopsis / Cultured plant cells, genes rpc00001