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: 4.62). 09/2005; 11(8):1291-302. DOI: 10.1261/rna.2060405
Source: PubMed

ABSTRACT 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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    • "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 · 4.62 Impact Factor
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    • "Mtr4p is an RNA helicase and is a subunit of the TRAMP complex, which is involved in RNA polyadenylation, directing the RNA for degradation by the exosome [29-31]. The Ski complex also contains a subunit with helicase activity, Ski2p, and is associated with the exosome in mRNA degradation [32,33]. The different protein cofactors that associate with the exosome may regulate its function. "
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    ABSTRACT: The archaeal exosome is formed by a hexameric RNase PH ring and three RNA binding subunits and has been shown to bind and degrade RNA in vitro. Despite extensive studies on the eukaryotic exosome and on the proteins interacting with this complex, little information is yet available on the identification and function of archaeal exosome regulatory factors. Here, we show that the proteins PaSBDS and PaNip7, which bind preferentially to poly-A and AU-rich RNAs, respectively, affect the Pyrococcus abyssi exosome activity in vitro. PaSBDS inhibits slightly degradation of a poly-rA substrate, while PaNip7 strongly inhibits the degradation of poly-A and poly-AU by the exosome. The exosome inhibition by PaNip7 appears to depend at least partially on its interaction with RNA, since mutants of PaNip7 that no longer bind RNA, inhibit the exosome less strongly. We also show that FITC-labeled PaNip7 associates with the exosome in the absence of substrate RNA. Given the high structural homology between the archaeal and eukaryotic proteins, the effect of archaeal Nip7 and SBDS on the exosome provides a model for an evolutionarily conserved exosome control mechanism.
    BMC Biochemistry 05/2010; 11:22. DOI:10.1186/1471-2091-11-22 · 1.94 Impact Factor
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