Volume 29, Issue 6, Pages 717-728 (March 2008) Structure of the Active Subunit of the Yeast Exosome Core, Rrp44: Diverse Modes of Substrate Recruitment in the RNase II Nuclease Family  Esben Lorentzen, Jerome Basquin, Rafal Tomecki, Andrzej Dziembowski, Elena Conti  Molecular Cell  Volume 29, Issue 6, Pages 717-728 (March 2008) DOI: 10.1016/j.molcel.2008.02.018 Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 1 Rrp44 Domain Structure and Contribution to Catalytic Activity (A) Schematic representation of the domain organization of eukaryotic Rrp44 (Dis3) and comparison with bacterial RNase II. The numbering refers to the S. cerevisiae and E. coli sequences, respectively. The two exoribonucleases feature a similar modular domain arrangement, with two cold shock domains (CSD1 and CSD2), a catalytic domain (RNB), and an S1 domain. In addition, Rrp44 has an N-terminal portion containing a predicted PIN domain that is not present in either RNase II or R. (B) S. cerevisiae Rrp44 full length and the N-terminal deletion mutant (Rrp44ΔN) have similar in vitro activities toward ssRNA substrates. Sequences of the substrates used in the RNA degradation experiments are shown in Table S1. The substrates (ss 17-A2, ss 17-A5, or ss 17-A14 oligoribonucleotides) were 5′ end labeled with [γ-32P]ATP, and the reaction products were analyzed by electrophoresis on a 20% acrylamide and 8 M urea gel, followed by phosphoimaging. Enzyme and substrate concentrations were 0.1 μM and 2 μM, respectively. They were incubated for 0, 5, 20, or 90 min, as indicated. The experiment with TAP-tagged yeast exosomes (purified either via Csl4 or Rrp44 affinity tags) containing Rrp44 and the other nine core subunits was performed as previously described (Dziembowski et al., 2007). (C) Activity of S. cerevisiae Rrp44 f.l. and ΔN as well as ten-subunit exosomes toward RNA duplexes. Reactions were done with ds 17-A5, ds 17-A7, or ds 17-A14 RNA substrates. Enzyme and substrate concentrations were 0.1 μM and 0.2 μM, respectively. Association with the exosome core considerably downregulates the activity of Rrp44 toward RNA duplexes. Molecular Cell 2008 29, 717-728DOI: (10.1016/j.molcel.2008.02.018) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 2 Structure of Rrp44ΔN in Complex with RNA View of the yeast Rrp44ΔN crystal structure in two orientations related by a 90° rotation about the vertical axis. The RNB domain (in blue) is flanked on one side by three OB-fold domains: the N-terminal CSD1 (in yellow) and CSD2 (in orange), and the C-terminal S1 (in red). Nine ordered nucleotides of a single-stranded poly(A)-RNA substrate (in black) are ordered in the structure. The most 3′ nucleotide is bound near a magnesium ion (in magenta) at the active site within the RNB domain. The 5′ end of the substrate is bound at the interface between the RNB and CSD1 domains. All ribbon drawings were carried out using PyMOL (DeLano, 2002, http://www.pymol.org). Molecular Cell 2008 29, 717-728DOI: (10.1016/j.molcel.2008.02.018) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 3 Rrp44 Sequence Conservation and Interactions The structure-based sequence alignment includes E. coli RNase II and RNase R as well as Rrp44 homologs from S. cerevisiae (Sc), H. sapiens (Hs), D. melanogaster (Dm) and T. brucei (Tb). The secondary structure elements of Sc Rrp44 are shown above the corresponding sequence (arrows for β strands and cylinders for α helices) and are colored in yellow (CSD1 domain), orange (CSD2), blue (RNB domain), and red (S1 domain). The β strands forming the OB fold of the CSD1, CSD2, and S1 domains are numbered β1–β5. Residues involved in binding the magnesium ion at the active site are highlighted in magenta, and residues involved in RNA binding are highlighted in green. Other conserved residues are highlighted in gray. Boxed in black are residues involved in interdomain interactions. Molecular Cell 2008 29, 717-728DOI: (10.1016/j.molcel.2008.02.018) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 4 Domain Configurations in the RNase II Family of Proteins (A) The structure of Rrp44 is shown on the left as in Figure 2, left panel. RNase II is shown on the right in the same orientation after optimal structural superposition of the RNB domains of the two structures. RNA is shown in black, and magnesium is shown in magenta. The lower panel shows a schematic representation of the domain orientations in the two structures. The β strands of the three OB folds are numbered (as in Figure 3) and have the typical Greek-key topology β3-β2-β1-β4-β5. Residues disordered in the long insertion between β4 and β5 of Rrp44 CSD1 are indicated with a dotted line. (B) Close-up of the intramolecular interactions between the CSD1 and CSD2 domains of Rrp44 (left panel) and of the interactions between the CSD1 and S1 domain (right panel). Residues at the interdomain interfaces are highlighted. Molecular Cell 2008 29, 717-728DOI: (10.1016/j.molcel.2008.02.018) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 5 RNA Binding to Rrp44 (A) View of the 9 nucleotides of poly(A) RNA bound to Rrp44 (numbered N1–N9). The unbiased Fo − Fc electron density map is contoured at 2.5 σ and is shown with the final model superimposed. (B) Schematic of the interactions at the RNA-binding site. The RNA nucleotides are numbered 1–9 from the 3′ end to the 5′ end. Residues of the RNB domain are boxed in blue, and residues of the CSD1 domain are boxed in yellow. Dotted lines represent polar interactions, and bold bars represent stacking interactions. The magnesium ion observed in the structure is shown in magenta. The empty magenta circle represents the position of a second magnesium ion, likely present in the wild-type protein (but missing in the present structure of the D551N mutant). (C) Close-up of the interactions between the 9 most 3′ nucleotides of ssRNA bound to either Rrp44 (left panel) or RNase II (right panel). The two structures are shown in a similar orientation after superimposition of the RNB domain. The nucleotides are labeled as in (A). A subset of residues involved in the interactions is highlighted. Molecular Cell 2008 29, 717-728DOI: (10.1016/j.molcel.2008.02.018) Copyright © 2008 Elsevier Inc. Terms and Conditions