SKI2 mediates degradation of RISC 5′-cleavage fragments and prevents secondary siRNA production from miRNA targets inArabidopsis

Abstract

Small regulatory RNAs are fundamental in eukaryotic and prokaryotic gene regulation. In plants, an important element of post-transcriptional control is effected by 20–24 nt microRNAs (miRNAs) and short interfering RNAs (siRNAs) bound to the ARGONAUTE1 (AGO1) protein in an RNA induced silencing complex (RISC). AGO1 may cleave target mRNAs with small RNA complementarity, but the fate of the resulting cleavage fragments remains incompletely understood. Here, we show that SKI2, SKI3 and SKI8, subunits of a cytoplasmic cofactor of the RNA exosome, are required for degradation of RISC 5′, but not 3′-cleavage fragments in Arabidopsis. In the absence of SKI2 activity, many miRNA targets produce siRNAs via the RNA-dependent RNA polymerase 6 (RDR6) pathway. These siRNAs are low-abundant, and map close to the cleavage site. In most cases, siRNAs were produced 5′ to the cleavage site, but several examples of 3′-spreading were also identified. These observations suggest that siRNAs do not simply derive from RDR6 action on stable 5′-cleavage fragments and hence that SKI2 has a direct role in limiting secondary siRNA production in addition to its function in mediating degradation of 5′-cleavage fragments.

Polysomes, Stress Granules, and Processing Bodies: A Dynamic Triumvirate Controlling Cytoplasmic mRNA Fate and Function

“…Alternatively, the uridylated fragments may be degraded in the 39 to 59 direction by the exosome (see below). This is supported by the observation of their increased abundance in genotypes of Arabidopsis deficient in exosome cofactor SUPER-KILLER (SKI) proteins (Branscheid et al, 2015). Recently, Arabidopsis RISC-INTERACTING CLEARING 39-59 EXORIBONUCLEASE1 (RICE1) and RICE2 were identified as AGO1-and AGO10-binding proteins that degrade uridylated RISC 59-cleaved fragments and facilitate RISC recycling (Zhang et al, 2017b).…”

The Slicer Activity of ARGONAUTE1 is Required Specifically for the Phasing, Not Production, of Trans-Acting Short Interfering RNAs in Arabidopsis

“…Fourth, two features of ectopic, miRNAtriggered secondary siRNAs in ski2 mutants pointed to a direct involvement of AGO1 in recruitment of RDR6. Although only RISC 59-cleavage fragments were stable in mutants of the exosome cofactor SUPERKILLER2, examples of miRNA targets with secondary siRNAs exclusively 39 to miRNA cleavage sites were identified (Branscheid et al, 2015). In addition, spreading of secondary siRNAs occurred on the cleavage fragment least stably base-paired to the miRNA, suggesting that RDR6 may be present at the AGO1-miRNA-target RNA complex prior to release of cleavage fragments and may use the cleavage fragment released first from RISC as template (Branscheid et al, 2015).…”

Antiviral Silencing and Suppression of Gene Silencing in Plants

“…RNA-DEPENDENT RNA POLYMER-ASES (RDRs, in Arabidopsis RDR1, 2, 3a, 3b, 3c and 6) (Wassenegger and Krczal 2006) protein recognize these molecules as their substrates and convert them into dsRNAs that enter/re-enter into silencing through DCL-mediated sRNA production. RDR6 is the main cytoplasmic enzyme to be involved in this process (Branscheid et al 2015;Martinez de Alba et al 2015;Mourrain et al 2000;Parent et al 2015b;Sijen et al 2001;Vaistij et al 2002;Voinnet et al 1998). Usage of RISC cleavage products by RDRs results in amplification of silencing response that may have also non-cell-autonomous consequences.…”