A complex prediction: three-dimensional model of the yeast exosome

Abstract

We present a model of the yeast exosome based on the bacterial degradosome component polynucleotide phosphorylase (PNPase). Electron microscopy shows the exosome to resemble PNPase but with key differences likely related to the position of RNA binding domains, and to the location of domains unique to the exosome. We use various techniques to reduce the many possible models of exosome subunits based on PNPase to just one. The model suggests numerous experiments to probe exosome function, particularly with respect to subunits making direct atomic contacts and conserved, possibly functional residues within the predicted central pore of the complex.

Fig. 1. Domain architectures for the exosome core and PNPase. Domains are taken from SMART (colored shapes) or Pfam (boxes). Regions of low sequence complexity are shown in pink.

Fig. 2. Alignment of RPDs. Residues are colored according to property conservation (red, polar; blue, small; yellow, hydrophobic), and numbers denote regions deleted for clarity. Boxes denote predicted functional site residues, and inverse characters those showing conservation across orthologs. Numbers below boxed residues denote FS-1 and FS-2 as shown in Figure 5. Species are abbreviated as follows: Sc, S. cerevisiae; Hs, H. sapiens; At, A. thaliana; Sp, S. pombe; Sa, Strepomyces antibioticus.

Fig. 3. EM and image processing of the exosome. (A) Micrograph of exosomes stained with uranyl acetate. (B) Image processing using the PNPase trimer (PDB code 1e3p) as initial reference: (1) different views of a surface representation of PNPase. This 3D map was used to generate the initial references by calculating 2D projections. (2) Projections of PNPase trimer shown in (1). Sixty-six of these projections equally distributed across the asymmetric unit were used as references for the initial alignment of exosome images and as anchor projections to determine spatial orientations of the class averages. (3) Class averages of the exosome images [direction of projection as in (2)]. (4) Surface representation of the 3D exosome map [views as in (1)]. (5) Projections of the map [same direction as in (2) and (3)]. (C) Exosome image processing without using a starting model. (1) Final class averages representing different views of the exosome. (2) Surface representation of the final 3D exosome map. (3) Projections of the map [directions as for (1)]. (D) Superposition of PNPase (solid surface) and the map of the exosome shown in (C) (wire frame). The view is towards the linker region of PNPase.

Fig. 4. Prediction of subunit arrangements for (A) DNA pol III β-subunit based on (B) PCNA-like structures. (C) Model of the exosome complex.

Fig. 5. Two views of a 3D model of the exosome core. Polar residues conserved across orthologs are labeled and correspond to inverse characters in Figure 2. Circles denote predicted functional sites (FS); boxed in Figure 2.