Domain interactions within the Ski2/3/8 complex and between the Ski complex and Ski7p

Section of Molecular Genetics and Microbiology, The University of Texas, Austin, TX 78712-0162, USA.
RNA (Impact Factor: 4.94). 09/2005; 11(8):1291-302. DOI: 10.1261/rna.2060405
Source: PubMed


The Ski complex (composed of Ski3p, Ski8p, and the DEVH ATPase Ski2p) is a central component of the 3'-5' cytoplasmic mRNA degradation pathway in yeast. Although the proteins of the complex interact with each other as well as with Ski7p to mediate degradation by exosome, a 3'-exonuclease complex, the nature of these interactions is not well understood. Here we explore interactions within the Ski complex and between the Ski complex and Ski7p using a directed two-hybrid approach combined with coimmunoprecipitation experiments. We also test the functional significance of these interactions in vivo. Our results suggest that within the Ski complex, Ski3p serves as a scaffold protein with its C terminus interacting with Ski8p, and the sub-C terminus interacting with Ski2p, while no direct interaction between Ski2p and Ski8p was found. Ski7p interacts with the Ski complex via its interaction with Ski8p and Ski3p. In addition, inactivating the Ski complex by mutating conserved residues in the DEVH helicase motif of Ski2 did not abrogate its interaction with Ski7p, indicating that Ski2p function is not necessary for this interaction.

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Available from: Arlen Johnson, Jan 13, 2014
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    • "Within this segment, helices H2 and H3 dock to the groove of TPRs 26–31, helix H4 binds in the groove of the adjacent TPRs 20– 25, and the b hairpin winds through TPRs 15–19. Consistently with the important role of the last superhelical turn of Ski3 in binding the anchor and inner segments of Ski2 in the structure, deletion of a region of Ski3 that with hindsight corresponds to TPRs 28–33 was shown to cause lethality in a yeast strain lacking XRN1 (Wang et al., 2005). The following RG segment of Ski2 (residues 129–164) lacks secondary structure elements but adopts a globular fold (Figure 3D). "
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    ABSTRACT: The Ski complex is a conserved multiprotein assembly required for the cytoplasmic functions of the exosome, including RNA turnover, surveillance, and interference. Ski2, Ski3, and Ski8 assemble in a tetramer with 1:1:2 stoichiometry. The crystal structure of an S. cerevisiae 370 kDa core complex shows that Ski3 forms an array of 33 TPR motifs organized in N-terminal and C-terminal arms. The C-terminal arm of Ski3 and the two Ski8 subunits position the helicase core of Ski2 centrally within the complex, enhancing RNA binding. The Ski3 N-terminal arm and the Ski2 insertion domain allosterically modulate the ATPase and helicase activities of the complex. Biochemical data suggest that the Ski complex can thread RNAs directly to the exosome, coupling the helicase and the exoribonuclease through a continuous RNA channel. Finally, we identify a Ski8-binding motif common to Ski3 and Spo11, rationalizing the moonlighting properties of Ski8 in mRNA decay and meiosis.
    Cell 08/2013; 154(4):814-26. DOI:10.1016/j.cell.2013.07.017 · 32.24 Impact Factor
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    • "Cells have therefore developed an elaborate system to eliminate nonstop transcripts via a pathway called nonstop decay (NSD) (Frischmeyer et al., 2002; van Hoof et al., 2002) and nonstop proteins via proteasomal degradation (Bengtson and Joazeiro, 2010). In the cytosol of Saccharomyces cerevisiae, nonstop mRNA is degraded mainly in the 3 0 / 5 0 direction by the cyotoplasmic exonuclease or exosome, which is recruited by Ski7 and functions in cooperation with the Ski complex containing the putative RNA helicase Ski2 (Frischmeyer et al., 2002; van Hoof et al., 2002; Araki et al., 2001; Wang et al., 2005). Cytosolic elimination of nonstop proteins from RNC complexes involves ubiquitination by the E3 ubiquitin ligase Ltn1 (Rkr1; Listerine in mammals) followed by proteasomal degradation (Bengtson and Joazeiro, 2010; Ito-Harashima et al., 2007). "
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    ABSTRACT: Because messenger RNAs without a stop codon (nonstop mRNAs) generate stalled ribosomes, cells have developed a mechanism allowing degradation of nonstop mRNAs and their translation products (nonstop proteins) in the cytosol. Here, we observe the fate of nonstop proteins destined for organelles such as the endoplasmic reticulum (ER) and mitochondria. Nonstop mRNAs for secretory-pathway proteins in yeast generate nonstop proteins that become stuck in the translocator, the Sec61 complex, in the ER membrane. These stuck nonstop secretory proteins avoid proteasomal degradation in the cytosol, but are instead released into the ER lumen through stalled ribosome and translocator channels by Dom34:Hbs1. We also found that nonstop mitochondrial proteins are cleared from the mitochondrial translocator, the TOM40 complex, by Dom34:Hbs1. Clearance of stuck nonstop proteins from organellar translocator channels is crucial for normal protein influx into organelles and for normal cell growth, especially when nonstop mRNA decay does not function efficiently.
    Cell Reports 09/2012; 2(3):447-53. DOI:10.1016/j.celrep.2012.08.010 · 8.36 Impact Factor
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    • "Removal of the insertion in Ski2 did not affect complex formation: Ski2-Dinsert comigrated with Ski3 and Ski8 in size-exclusion chromatography (Fig. 4A). We next tested whether the insertion of Ski2 is required to bind Ski7, an outer-layer protein of the Ski complex that mediates the interaction with the exosome (Araki et al. 2001; Wang et al. 2005). In in-vitro pull-down experiments with purified proteins, f.l. "
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    ABSTRACT: Ski2 is a cytoplasmic RNA helicase that functions together with the exosome in the turnover and quality control of mRNAs. Ski2 is conserved in eukaryotes and is related to the helicase Mtr4, a cofactor of the nuclear exosome involved in the processing and quality control of a variety of structured RNAs. We have determined the 2.4 Å resolution crystal structure of the 113 kDa helicase region of Saccharomyces cerevisiae Ski2. The structure shows that Ski2 has an overall architecture similar to that of Mtr4, with a core DExH region and an extended insertion domain. The insertion is not required for the formation of the Ski2-Ski3-Ski8 complex, but is instead an RNA-binding domain. While this is reminiscent of the Mtr4 insertion, there are specific structural and biochemical differences between the two helicases. The insertion of yeast Mtr4 consists of a β-barrel domain that is flexibly attached to a helical stalk, contains a KOW signature motif, and binds in vitro-transcribed tRNA(i)(Met), but not single-stranded RNA. The β-barrel domain of yeast Ski2 does not contain a KOW motif and is tightly packed against the helical stalk, forming a single structural unit maintained by a zinc-binding site. Biochemically, the Ski2 insertion has broad substrate specificity, binding both single-stranded and double-stranded RNAs. We speculate that the Ski2 and Mtr4 insertion domains have evolved with different properties tailored to the type of transcripts that are the substrates of the cytoplasmic and nuclear exosome.
    RNA 11/2011; 18(1):124-34. DOI:10.1261/rna.029553.111 · 4.94 Impact Factor
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