Sequencing of RDR6-dependent double-stranded RNAs reveals novel features of plant siRNA biogenesis

Abstract

Biogenesis of trans-acting siRNAs (tasiRNAs) is initiated by miRNA-directed cleavage of TAS gene transcripts and requires RNA-dependent RNA polymerase 6 (RDR6) and Dicer-like 4 (DCL4). Here, we show that following miR173 cleavage the entire polyadenylated parts of Arabidopsis TAS1a/b/c and TAS2 transcripts are converted by RDR6 to double-stranded (ds)RNAs. Additionally, shorter dsRNAs are produced following a second cleavage directed by a TAS1c-derived siRNA. This tasiRNA and miR173 guide Argonaute 1 complexes to excise the segments from TAS2 and three TAS1 transcripts including TAS1c itself to be converted to dsRNAs, which restricts siRNA production to a region between the two cleavage sites. TAS1c is also feedback regulated by a cis-acting siRNA. We conclude that TAS1c generates a master siRNA that controls a complex network of TAS1/TAS2 siRNA biogenesis and gene regulation. TAS1/TAS2 short dsRNAs produced in this network are processed by DCL4 from both ends in distinct registers, which increases repertoires of tasiRNAs.

Novel Stress-Inducible Antisense RNAs of Protein-Coding Loci Are Synthesized by RNA-Dependent RNA Polymerase

“…S8C). These observations suggest that most of the dsRNAs did not accumulate as intermediates of small RNA synthesis, because they were quickly processed into small RNAs (Rajeswaran et al, 2012). The RDR1/2/6-dependent antisense RNA loci were also compared with the specific small RNA loci reported previously (Martínez de Alba et al, 2015;Zhang et al, 2015).…”

“…Two RDR1/2/6-dependent antisense RNAs that were mapped on TAS1B existed upstream of a major small RNA-generated region (Supplemental Fig. S8A, Rajeswaran et al, 2012). In addition, we tested whether accumulation of sense and antisense RNAs was reduced through the small RNA production process on Figure 4.…”

“…The increased levels of uncapped sense-stranded RNAs in these mutants were used as templates to produce dsRNAs during the PTGS process. The dsRNAs were synthesized by RNA-dependent RNA polymerase 6 (RDR6), one of six Arabidopsis RDRs (Rajeswaran et al, 2012;Martínez de Alba et al, 2015;Zhang et al, 2015), and processed into 21-22 nt siRNA by DCL4 (Zhang et al, 2015). Another possible mechanism of antisense RNA synthesis is transcriptional gene silencing (TGS) of related RNAs that are synthesized by a DNA-directed RNA polymerase IV (PolIV) and RDR2 (Xie et al, 2004;Swiezewski et al, 2009).…”

New Generation of Artificial MicroRNA and Synthetic Trans-Acting Small Interfering RNA Vectors for Efficient Gene Silencing in Arabidopsis

“…It is possible that one or more factors in the AtTAS1c-dependent tasiRNA-generating pathway is limiting relative to the ubiquitous miRNA biogenesis factors. It is also possible that RDR6-dependent TAS1c dsRNAs may be processed by DCL4 from both ends, resulting in the production of tasiRNAs in two registers (Rajeswaran et al, 2012) and limiting the accumulation of accurately processed syn-tasiRNAs from positions D3[+] and D4 [+].…”

An endogene-resembling transgene is resistant to DNA methylation and systemic silencing

“…Thus, while initial cleavage is conducted by primary siRNAs, RDR6 transcription of the cleavage products generates secondary dsRNA that is processed into secondary siRNAs. 19,20 In contrast to primary siRNAs, that match only to a sub-region of the target RNA, secondary siRNAs may map upstream and downstream from the binding sites of the primary siRNAs. This spreading of silencing beyond initial target sites is termed transitivity.…”