RNA channelling by the archaeal exosome

Abstract

Exosomes are complexes containing 3' --> 5' exoribonucleases that have important roles in processing, decay and quality control of various RNA molecules. Archaeal exosomes consist of a hexameric core of three active RNase PH subunits (ribosomal RNA processing factor (Rrp)41) and three inactive RNase PH subunits (Rrp42). A trimeric ring of subunits with putative RNA-binding domains (Rrp4/cep1 synthetic lethality (Csl)4) is positioned on top of the hexamer on the opposite side to the RNA degrading sites. Here, we present the 1.6 A resolution crystal structure of the nine-subunit exosome of Sulfolobus solfataricus and the 2.3 A structure of this complex bound to an RNA substrate designed to be partly trimmed rather than completely degraded. The RNA binds both at the active site on one side of the molecule and on the opposite side in the narrowest constriction of the central channel. Multiple substrate-binding sites and the entrapment of the substrate in the central channel provide a rationale for the processive degradation of extended RNAs and the stalling of structured RNAs.

Figure 1

Structure of the complete 270 kDa Sulfolobus solfataricus exosome. (A) Two views of the complex, with Rrp41 in blue, Rrp42 in green and Rrp4 in yellow. Manganese ions are shown in cyan. The views are rotated by 90° around the horizontal axis. This figure and all others representing structures were generated with the program PYMOL (http://pymol.sourceforge.net, Warren L. DeLano). (B) View of the S. solfataricus exosome (Rrp4 in yellow, and Rrp41 and Rrp42 in light grey) superposed on the A. fulgidus exosome using the RNase PH cores (Rrp4 in red, Rrp41 and Rrp42 in dark grey). The structures are viewed as in (A), left. The three domains of Rrp4 (N-terminal, S1 and KH) are indicated. (C) View of the S. solfataricus exosome superposed on the human exosome using the RNase PH cores (light grey for S. solfataricus and dark grey for human RNase PH). Ss-Rrp4 is shown in yellow, Hs-Rrp40 in magenta, Hs-Rrp4 in green and Hs-Csl4 in pink. Csl4, cep1 synthetic lethality; Hs, Homo sapiens; Rrp, ribosomal RNA processing factor; Ss, Sulfolobus solfatarious.

Figure 2

RNA binding in the central channel of the archaeal exosome. (A) An overview showing the electron density of the ribonucleotide bound at the entrance of the channel (2.3 Å resolution F_o−F_c map, contoured at 2σ) with the final model superimposed. The complex crystallized with one unique heterotrimer in the asymmetric unit, with the full complex generated by crystallographic threefold symmetry (indicated by the triangular symbol). The map is unbiased as it is calculated using model phases before any ligands were introduced. (B) Close-up of the density and model at the entrance of the channel. The loop of Rrp41 (residues 62–70) is shown in cyan forming the constriction where the ribonucleotide binds. Schematics of the ligands used for the soaking experiments are shown below the pictures. (C) Anomalous electron density map showing the binding of tungstate at the narrow constriction of the central channel. The map is calculated to 4 Å resolution (no useful anomalous signal is present beyond 4 Å), contoured at 4σ and is calculated using model phases after restrained refinement against all data extending to 2.5 Å resolution. (D) Similar view for the anomalous electron density obtained by soaking a structured RNA substrate with an iodinated 3′ poly(U) tail. The anomalous map is calculated as in (C). Rrp, ribosomal RNA processing factor.

Figure 3

Metal ions mediate subunit interactions. (A) A close-up view showing the structural manganese (Mn) ion-binding site in the Sulfolobus solfataricus (Ss) exosome, located at the interface between Ss-Rrp41 (blue) and Ss-Rrp4 (yellow). A 2.4 Å resolution 2F_o−F_c electron density map contoured at 1σ is shown in blue and an anomalous map at 4 Å resolution contoured at 4σ is shown in magenta. Residues that coordinate the metal ion are labelled. (B) The equivalent region of the Archaeglobus fulgidus exosome structure showing that the metal ion-binding site found in the S. solfataricus exosome is replaced by a direct salt-bridge (dotted line). (C) Sequence alignment showing that the Rrp41–Rrp4 contacts are probably mediated either by divalent metal ions or by a direct salt bridge in exosomes from different organisms. Sequences included are: S. solfataricus (SULSO), Pyrococcus furiosus (PYRFU), Dictyostelium discoideum (DICDI), A. fulgidus (ARCFU), Saccharomyces cerevisiae (YEAST) and Homo sapiens (HUMAN). Numbers in parentheses denote overall percentage identity of the full-length proteins to the S. solfataricus sequence. D, aspartic acid; E, glutamic acid; K, lysine; Rrp, ribosomal RNA processing factor.

Figure 4

Ribbon representation of the Sulfolobus solfataricus exosome including a Corey–Pauling–Koltun model of the RNA bound in the structure. A possible path for RNA substrates is indicated by a magenta-coloured line. In this model, the 3′ end of the RNA substrate is recruited by the S1 pore of the Rrp4/Csl4 cap and then threaded through the neck of the central channel to the processing chamber, where it binds at one of the active sites and is degraded in a processive manner. Csl4, cep1 synthetic lethality; Rrp, ribosomal RNA processing factor.