Context-Dependent Dual Role of SKI8 Homologs in mRNA Synthesis and Turnover

Abstract

Eukaryotic mRNA transcription and turnover is controlled by an enzymatic machinery that includes RNA polymerase II and the 3′ to 5′ exosome. The activity of these protein complexes is modulated by additional factors, such as the nuclear RNA polymerase II-associated factor 1 (Paf1c) and the cytoplasmic Superkiller (SKI) complex, respectively. Their components are conserved across uni- as well as multi-cellular organisms, including yeast, Arabidopsis, and humans. Among them, SKI8 displays multiple facets on top of its cytoplasmic role in the SKI complex. For instance, nuclear yeast ScSKI8 has an additional function in meiotic recombination, whereas nuclear human hSKI8 (unlike ScSKI8) associates with Paf1c. The Arabidopsis SKI8 homolog VERNALIZATION INDEPENDENT 3 (VIP3) has been found in Paf1c as well; however, whether it also has a role in the SKI complex remains obscure so far. We found that transgenic VIP3-GFP, which complements a novel vip3 mutant allele, localizes to both nucleus and cytoplasm. Consistently, biochemical analyses suggest that VIP3–GFP associates with the SKI complex. A role of VIP3 in the turnover of nuclear encoded mRNAs is supported by random-primed RNA sequencing of wild-type and vip3 seedlings, which indicates mRNA stabilization in vip3. Another SKI subunit homolog mutant, ski2, displays a dwarf phenotype similar to vip3. However, unlike vip3, it displays neither early flowering nor flower development phenotypes, suggesting that the latter reflect VIP3's role in Paf1c. Surprisingly then, transgenic ScSKI8 rescued all aspects of the vip3 phenotype, suggesting that the dual role of SKI8 depends on species-specific cellular context.

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

“…Both ski8-7 and to a lesser extent ski8-6 display dwarf and bushy phenotypes (Fig. 7, A and B), and the ski8-7 mutation also results in flower abnormalities (Dorcey et al, 2012) absent from ski8-6. When ski8-6 and ski8-7 were crossed into the cer7-3 background, the resulting double mutants showed dramatically improved wax accumulation on inflorescence stems compared with cer7-3 and substantially restored CER3 transcript levels (Fig.…”

“…3A) and AtSKI2 (At3g46960), which encodes the RNA helicase subunit of the SKI complex (Jolivet et al, 2006;Dorcey et al, 2012). Because the SKI complex is conserved across many uni-and multicellular organisms, including yeast, Drosophila, Arabidopsis, and humans, we suspected that mutations in AtSKI2, just like those in AtSKI3, might also be able to restore the wax phenotype of the cer7 mutant.…”

“…In contrast, the N terminus of ScSKI2 that is necessary and sufficient for interaction with ScSKI3 and ScSKI8 in yeast does not exhibit significant homology with the Arabidopsis AtSKI2. This does not, however, preclude successful interactions of AtSKI2 with AtSKI3 and AtSKI8 subunits as demonstrated by pull-down experiments in Arabidopsis (Dorcey et al, 2012;Zhang et al, 2015). It is well-documented that a third component of the SKI complex, named SKI8, is indispensable for the cytoplasmic functions of the exosome (Brown et al, 2000;Orban and Izaurralde, 2005;Dorcey et al, 2012).…”

“…This does not, however, preclude successful interactions of AtSKI2 with AtSKI3 and AtSKI8 subunits as demonstrated by pull-down experiments in Arabidopsis (Dorcey et al, 2012;Zhang et al, 2015). It is well-documented that a third component of the SKI complex, named SKI8, is indispensable for the cytoplasmic functions of the exosome (Brown et al, 2000;Orban and Izaurralde, 2005;Dorcey et al, 2012). To determine whether AtSKI8 works together with AtSKI2 and AtSKI3 to regulate stem wax biosynthesis in Arabidopsis, we obtained two T-DNA insertional mutants in the AtSKI8 gene from the Arabidopsis Biological Resource Center and employed them for reverse genetic analyses.…”

“…Positional cloning and analysis of WAR1 and WAR7 genes indicated that they carry mutations in Arabidopsis SUPERKILLER3 (AtSKI3; At1g76630) and AtSKI2 (At3g46960), respectively, that encode components of the SKI complex. In Arabidopsis, as in other eukaryotes studied thus far, SKI proteins have been shown to be required for cytoplasmic 39-to-59 RNA degradation by the exosome (Houseley et al, 2006;Dorcey et al, 2012;Halbach et al, 2013;Lange et al, 2014;Zhang et al, 2015), but evidence for their functional association into a complex is missing. Detailed analyses of the phenotypes of the ski single and ski cer7 double mutants reported here demonstrate that all SKI proteins participate in the CER7/ RRP45B-mediated control of wax deposition in developing Arabidopsis inflorescence stems, and support the notion that they act in the same pathway or complex.…”