Abstract
The RNA exosome is an evolutionarily conserved multi-protein complex involved in the 3' degradation of a variety of RNA transcripts. In the nucleus, the exosome participates in the maturation of structured RNAs, in the surveillance of pre-mRNAs and in the decay of a variety of noncoding transcripts. In the cytoplasm, the exosome degrades mRNAs in constitutive and regulated turnover pathways. Several structures of subcomplexes of eukaryotic exosomes or related prokaryotic exosome-like complexes are known, but how the complete assembly is organized to fulfil processive RNA degradation has been unclear. An atomic snapshot of a Saccharomyces cerevisiae 420 kDa exosome complex bound to an RNA substrate in the pre-cleavage state of a hydrolytic reaction has been determined. Here, the crystallographic steps towards the structural elucidation, which was carried out by molecular replacement, are presented.
Highlights
The eukaryotic exosome core, Exo-9, contains six RNase PHlike subunits that assemble into a ring-like structure and three proteins composed of S1/KH domains forming a coaxial ring (Fig. 1, top; Mitchell et al, 1997)
PNPase consists of three identical proteins, each containing two RNase PH domains and an S1/KH domain in a single polypeptide chain (Symmons et al, 2000)
The archaeal structure was at significantly higher resolution and was more accurate and, with hindsight, was more similar at the tertiary-structure level
Summary
The eukaryotic exosome core, Exo-9, contains six RNase PHlike subunits that assemble into a ring-like structure and three proteins composed of S1/KH domains (so-called cap proteins) forming a coaxial ring (Fig. 1, top; Mitchell et al, 1997). Two distinct proteins (Rrp and Rrp42) with an RNase PH fold and a third protein (Rrp or Csl4) with S1/KH domains trimerize into an Exo-9-like architecture (Buttner et al, 2005; Lorentzen et al, 2005; Navarro et al, 2008) Both complexes present phosphorolytic ribonuclease activities provided by one of the RNase PH subunits. A first view of how the processive nuclease might bind Exo-9 was later provided by the structure of Rrp bound to two RNase PH-like proteins of the yeast exosome (Rrp and Rrp45; Bonneau et al, 2009).
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