The exosome complex is the major eukaryotic 3' --> 5' exonuclease involved in several RNA surveillance, turnover and processing pathways both in the nucleus and cytoplasm. The conserved core of the exosome contains nine subunits that form a ring similar to the phosphorolytic bacterial PNPase and archaeal exosome, as well as Dis3. Dis3 is homologous to bacterial RNase II, a hydrolytic enzyme. Previous studies have suggested that all subunits are active 3' --> 5' exoRNases. We have shown that Dis3 is responsible for exosome core activity (Dziembowski et al., 2007). The purified exosome core has a hydrolytic, processive and Mg(2+)-dependent activity with characteristics similar to those of recombinant Dis3. Moreover, a catalytically inactive Dis3 mutant has no exosome core activity in vitro and shows in vivo RNA degradation phenotypes similar to those resulting from exosome depletion. In contrast, mutations in Rrp41, the only subunit carrying a conserved phosphorolytic site, appear phenotypically identical to wild-type yeast. We observed that the yeast exosome ring mediates interactions with protein partners, providing an explanation for its essential function. It was suggested that exosome has a similar way of action as the proteasome, where ring structure restricts access of the substrates (Lorentzen and Conti, 2006), however, the observation that Dis3p is responsible for core exosome activity makes this less likely. Furthermore, the recently solved structures of archaeal exosome-like complexes, and the human 9-subunit complex does not describe the mechanism of action of the eukaryotic exosome because Dis3 is absent in these structures (Buttner et al., 2005; Lorentzen and Conti, 2005; Lorentzen et al., 2007; Liu et al., 2006). The RNA path to reach the exosome active site is not known. In addition the mechanism of exosome activation by associated factors is also not well understood. We clearly need more biochemical and structural data to understand how the exosome can participate in so many diverse functions and how these activities are tightly regulated. Currently, as our attempts to crystallize 10 subunit exosome complexes were unsuccessful, we are combining low resolution structural data with comparative structural modelling to derive a semi atomic model of this complex. The exosome subunit interaction map obtained by native Mass Spectromtry will be very helpful in that (Hernandez and Dziembowski et al., 2006). We are also trying to understand the mechanism of activation of the exosome by its associated complexes by combining biochemical and structural biology approaches.

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