Structure of a Cytoplasmic 11-Subunit RNA Exosome Complex

摘要

class="head no_bottom_margin" id="sec1title">IntroductionThe eukaryotic RNA exosome is a conserved ribonuclease complex that controls the quantity and quality of a large number of RNAs. Exosome-mediated RNA degradation leads to the elimination of nuclear and cytoplasmic transcripts in turnover and quality control pathways or to partial trimming of RNA precursors in processing pathways (reviewed in , , , , , ). The core complex of the RNA exosome was originally discovered from genetic and biochemical analyses in budding yeast () and has since been characterized at the molecular and structural level (reviewed in , ). Orthologs have also been identified in other eukaryotes and have been linked to Mendelian diseases in the human population (, ).The yeast exosome core complex is formed by ten different proteins. Only a single subunit (Rrp44, also known as Dis3) is catalytically active (, ). The other nine core subunits (Exo9) form a cylindrical structure that threads RNA substrates to the Rrp44 exoribonuclease site (, , href="#bib34" rid="bib34" class=" bibr popnode">Makino et al., 2013, href="#bib56" rid="bib56" class=" bibr popnode">Wasmuth et al., 2014). Yeast Exo10 is present in the nucleus as well as in the cytoplasm but binds compartment-specific cofactors. In the nucleus, the exosome associates with an additional ribonuclease complex (Rrp6-Rrp47), an RNA helicase (Mtr4), and a small protein (Mpp6) to form a 14-subunit assembly (href="#bib8" rid="bib8" class=" bibr popnode">Butler and Mitchell, 2010, href="#bib45" rid="bib45" class=" bibr popnode">Schuch et al., 2014). In the cytoplasm, the exosome functions together with the Ski2-Ski3-Ski8-Ski8 (Ski) complex, a tetrameric assembly centered at an Mtr4-like RNA helicase (Ski2) (href="#bib3" rid="bib3" class=" bibr popnode">Anderson and Parker, 1998, href="#bib7" rid="bib7" class=" bibr popnode">Brown et al., 2000, href="#bib21" rid="bib21" class=" bibr popnode">Halbach et al., 2013). Orthologs of these exosome cofactors are well conserved in eukaryotes (href="#bib43" rid="bib43" class=" bibr popnode">Schilders et al., 2007, href="#bib8" rid="bib8" class=" bibr popnode">Butler and Mitchell, 2010, href="#bib42" rid="bib42" class=" bibr popnode">Schaeffer et al., 2011), and several are mutated in human diseases (reviewed in href="#bib17" rid="bib17" class=" bibr popnode">Fabre and Badens, 2014, href="#bib49" rid="bib49" class=" bibr popnode">Staals and Pruijn, 2010).An additional cofactor, Ski7, bridges the interaction between the exosome and Ski complexes in S. cerevisiae (href="#bib4" rid="bib4" class=" bibr popnode">Araki et al., 2001, href="#bib21" rid="bib21" class=" bibr popnode">Halbach et al., 2013, href="#bib52" rid="bib52" class=" bibr popnode">van Hoof et al., 2000, href="#bib54" rid="bib54" class=" bibr popnode">Wang et al., 2005). The N-terminal exosome-binding and Ski-binding domains of Ski7 are required for all known exosome functions in the cytoplasm, including mRNA turnover and quality-control pathways (href="#bib4" rid="bib4" class=" bibr popnode">Araki et al., 2001, href="#bib42" rid="bib42" class=" bibr popnode">Schaeffer et al., 2011, href="#bib53" rid="bib53" class=" bibr popnode">van Hoof et al., 2002. The C-terminal GTPase-like domain of Ski7 has instead a specific role in nonstop decay (NSD) (href="#bib53" rid="bib53" class=" bibr popnode">van Hoof et al., 2002). NSD is one of the quality-control pathways that monitors the process of mRNA translation: it eliminates defective transcripts where the absence of in-frame termination codons causes ribosomes to stall upon translating the 3′ poly(A) tail (reviewed in href="#bib23" rid="bib23" class=" bibr popnode">Inada, 2013, href="#bib26" rid="bib26" class=" bibr popnode">Klauer and van Hoof, 2012, href="#bib32" rid="bib32" class=" bibr popnode">Lykke-Andersen and Bennett, 2014, href="#bib47" rid="bib47" class=" bibr popnode">Shoemaker and Green, 2012). S. cerevisiae Ski7 is a paralogue of the ribosome recycling factor Hbs1. Hbs1 functions in no-go decay (NGD), another translational quality-control pathway that targets and degrades transcripts with ribosomes stalled in the coding region or in the 3′ untranslated region (href="#bib13" rid="bib13" class=" bibr popnode">Doma and Parker, 2006, href="#bib20" rid="bib20" class=" bibr popnode">Guydosh and Green, 2014; and reviewed in href="#bib23" rid="bib23" class=" bibr popnode">Inada, 2013, href="#bib32" rid="bib32" class=" bibr popnode">Lykke-Andersen and Bennett, 2014, href="#bib47" rid="bib47" class=" bibr popnode">Shoemaker and Green, 2012). Yeast Hbs1 and Dom34 have also been recently implicated in NSD (href="#bib51" rid="bib51" class=" bibr popnode">Tsuboi et al., 2012).The SKI7 and HBS1 paralogous genes originated from an ancestral genome duplication event in budding yeast (href="#bib36" rid="bib36" class=" bibr popnode">Marshall et al., 2013). The Ski7 and Hbs1 proteins comprise a similar translational GTPase-like domain (href="#bib27" rid="bib27" class=" bibr popnode">Kowalinski et al., 2015) and are expected to share similarities in recognizing stalled ribosomes (href="#bib53" rid="bib53" class=" bibr popnode">van Hoof et al., 2002). However, they diverge in their activities (GTP binding versus GTP hydrolysis), mRNA targets (nonstop versus no-go mRNAs), and interacting proteins (exosome-Ski versus Dom34). Another conspicuous difference is that Hbs1 is conserved across eukaryotes while Ski7 orthologs have only been identified in a subset of fungal species (href="#bib36" rid="bib36" class=" bibr popnode">Marshall et al., 2013). Although Ski7 is currently assumed to be a specialized protein in yeasts, several observations argue against such evolutionary restriction. First, given that the exosome and Ski complexes are conserved from yeast to human, it is reasonable to expect that a Ski7-like cofactor would bridge their interaction also in higher eukaryotes. Second, NSD is not limited to yeast but also exists in mammalian cells, where it depends on HBS1-DOM34 as well as exosome and SKI proteins (href="#bib41" rid="bib41" class=" bibr popnode">Saito et al., 2013). In this work, we set out to visualize how the yeast exosome interacts with Ski7, and based on the structural information, we identified isoform 3 of HBS1L as the human counterpart of Ski7.