SUPERKILLER complex components are required for the RNA exosome-mediated control of cuticular wax biosynthesis in Arabidopsis inflorescence stems

Abstract

ECERIFERUM7 (CER7)/AtRRP45B core subunit of the exosome, the main cellular 39-to-59 exoribonuclease, is a positive regulator of cuticular wax biosynthesis in Arabidopsis (Arabidopsis thaliana) inflorescence stems. CER7-dependent exosome activity determines stem wax load by controlling transcript levels of the wax-related gene CER3. Characterization of the second-site suppressors of the cer7 mutant revealed that small interfering RNAs (siRNAs) are direct effectors of CER3 expression. To explore the relationship between the exosome and posttranscriptional gene silencing (PTGS) in regulating CER3 transcript levels, we investigated two additional suppressor mutants, wax restorer1 (war1) and war7. We show that WAR1 and WAR7 encode Arabidopsis SUPERKILLER3 (AtSKI3) and AtSKI2, respectively, components of the SKI complex that associates with the exosome during cytoplasmic 39-to-59 RNA degradation, and that CER7-dependent regulation of wax biosynthesis also requires participation of AtSKI8. Our study further reveals that it is the impairment of the exosome-mediated 39-59 decay of CER3 transcript in the cer7 mutant that triggers extensive production of siRNAs and efficient PTGS of CER3. This identifies PTGS as a general mechanism for eliminating highly abundant endogenous transcripts that is activated when 39-to-59 mRNA turnover by the exosome is disrupted. Diminished efficiency of PTGS in ski mutants compared with cer7, as evidenced by lower accumulation of CER3-related siRNAs, suggests that reduced amounts of CER3 transcript are available for siRNA synthesis, possibly because CER3 mRNA that does not interact with SKI is degraded by 59-to-39 XRN4 exoribonuclease.

Subcellular Compartmentation of Alternatively Spliced Transcripts Defines SERINE/ARGININE-RICH PROTEIN30 Expression

“…The following mutants have been used in different experiments: sr30 (GK325-E11, N322146), lba1 (Yoine et al, 2006), upf3-1 (Hori and Watanabe, 2005), 35S: HF-RPL18 (N66056; Zanetti et al, 2005), sop2-1 (Hématy et al, 2016), mtr3 (also referred to as rrp41l; Yang et al, 2013), hen2-4 and mtr4-1 (Lange et al, 2011), ski2-6 (Zhao andKunst, 2016), xrn4-5 (Souret et al, 2004), and xrn2-1 xrn4-6, xrn3-3 xrn4-6, and fry1-6 (Gy et al, 2007). The following genes have been analyzed: AT1G09140 (SR30), AT2G37340 (RS2Z33), AT5G37055 (SEF), AT1G13320 (PP2A), and At5G09810 (ACTIN7).…”

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

“…Disruption of AtSKI8 function results in the stabilization of mRNAs targeted for degradation by the exosome (Dorcey et al, 2012). Similar to the exosome core components RRP45A and RRP45B, all three SKI proteins are required for the exosomal degradation of the AtECERIFERUM3 transcript during stem wax deposition (Zhao and Kunst, 2016).…”

“…Arabidopsis SKI2, SKI3, and SKI8 form a stable complex in vivo, with SKI2 and SKI3 also present in cytoplasmic foci (Dorcey et al, 2012;Zhang et al, 2015;Zhao and Kunst, 2016). Disruption of AtSKI8 function results in the stabilization of mRNAs targeted for degradation by the exosome (Dorcey et al, 2012).…”

RST1 and RIPR connect the cytosolic RNA exosome to the Ski complex inArabidopsis

“…1A,B). Previous studies established that the wax deficiency of cer7 mutants is due to post-transcriptional silencing of the mRNA encoding CER3 43–45,48 , a subunit of a VLC alkane-forming complex 46,47 . Indeed, RNA blots revealed a severe reduction of the CER3 mRNA and an accumulation of CER3 -derived small RNAs in both cer7 and rst1 mutants (Fig 1C).…”

“…This and the identification of small RNAs accumulating in cer7 mutants revealed that the wax deficiency observed in cer7 plants is due to post-transcriptional silencing of CER3 mRNAs 44,45 , encoding a protein that together with the aldehyde decarbonylase CER1 catalyses the synthesis of VLC alkanes from VLC acyl-CoAs 46,47 . These results demonstrated that the RNA exosome contributes to the degradation of the CER3 mRNA and that the wax-deficient phenotype of cer7 mutants is a consequence of the established link between RNA degradation and silencing pathways 44,45,48 . Indeed, in plants, the two major exoribonucleolytic pathways that degrade RNAs either from 5’ to 3’ or from 3’ to 5’ also prevent that degradation intermediates such as uncapped or RISC-cleaved mRNAs are attacked by post-transcriptional gene silencing (PTGS) 49–54 , a mechanism required for the destruction of non-self RNAs originating from viruses or transgenes 55 .…”