Overall sequence conservation and structure of the PIN domain of hSMG6 (also known as EST1A) and hSMG5 (also known as EST1B). (A) Structure-based sequence alignment of SMG6-PIN and SMG5-PIN with related proteins of known structure: the archaeal PIN domains (pdb code 1o4w and 1w8i) and the nuclease domains of T4 RNase H (RNH_T4) and Thermus aquaticus TAQ polymerase (TAQ_ta). The secondary structure elements of human hSMG6 and human hSMG5 are indicated (above and below the sequences, respectively) with the symbols h for α-helices, b for β-strands, dots for extended portions and no symbol for residues that are disordered and are not present in the final model. Asterisks indicate residues of human hSMG6 present at equivalent structural position in the PIN domains of known structure (i.e. r.m.s.d. less than 2.5 Å between corresponding Cα atoms after optimal superposition). The three aspartic-acid residues at the active site of nucleases are highlighted in red. Highlighted in blue are residues that are conserved in SMG6 based on a sequence alignment including SMG6 from Homo sapiens (hs) and Drosophilla melanogaster (dm) (shown) and also Caenorhabditis elegans and Danio rerio (not shown). Highlighted in green are conserved residues of SMG5 as of a sequence alignment using SMG5 sequences from the same species. (B) Structure of human SMG6-PIN (residues 1239–1417) in two orientations related by a 90° rotation along a horizontal axis. Helices are shown in blue, β-strands in gray and disordered parts of the polypeptide chain are indicated with dots. In sticks are the three conserved aspartic acids that are highlighted in red in (A). The N- and C-termini as well as the aspartates are labeled. All ribbon drawings in the figures were prepared using CCP4 mg (Potterton et al, 2002). (C) Structure of human SMG5-PIN (residues 855–1013). The structure is shown in similar orientations to the hSMG6-PIN structure in (B), with analogous labeling and color coding (with exception of the helices that are shown in green).

The PIN domains of human SMG6 and SMG5 have a core structure similar to 5′ nucleases. (A, B) Structural superposition of hSMG6-PIN (blue) to an archaeal PIN domain from Archaeoglobus fulgidus (pdb code 1o4w, in pink). The structures are viewed in the same orientation as the left panels in Figure 1B. (B) A detailed view of the active site region. The conserved aspartic acid residues (in red in Figure 1A) are shown in stick format and labeled with residue numbers from both proteins. (C, D) Structural superposition of hSMG6-PIN (blue) to T4 RNase H (in orange), in a similar representation to panels (A, B). The gray sphere in the right panel represent an Mg ion bound in the T4 RNase H structure. (E, F) Structural superposition of hSMG6-PIN (blue) to hSMG5-PIN (green), in a similar representation to panels (A, B). The superpositions show that hSMG6-PIN has a similar active site as compared to structurally homologous nucleases, while hSMG5-PIN shares only partial conservation.

The PIN domain of SMG6 has nuclease activity on single-stranded RNA. (A) hSMG6-PIN and hSMG5-PIN (either wild-type or mutant) were incubated with a ³²P-labelled (U)₃₀ RNA with or without addition of 3 mM MgCl₂, 3 mM MnCl₂ or 2.5 mM EDTA as indicated. One eight of the input was loaded immediately after mixing (time 0, RNA loading control) and one eight after incubation at room temperature for 150 min on 10% polyacrylamide gel containing 8 M urea. RNA signals were visualized by autoradiography and quantified using the Fuji FLA-2000 phosphoimager system. The lower panel shows the RNA signals measured following the 150-min incubation normalized to those at time 0. Proteins used in (A) were analyzed by SDS–PAGE, followed by Coomassie staining (protein loading control). (B) Pattern of decay intermediates. 600 ng of cold (U)₃₀ RNA (10.8 pmol) were mixed with either 6 ng of 5′-[³²P]-end-labelled (U)₃₀ RNA (0.1 pmol) or 6 ng of 3′-[³²P]-end-labelled (U)₃₀ RNA (0.1 pmol) and incubated with 5 μg of purified proteins in 60 μl of buffer containing 20 mM HEPES pH 7.5, 150 mM NaCl, 10% glycerol and 1 mM DTT. Samples were analyzed at the indicated time points. The extent of RNA degradation was evaluated by analyzing the reaction mixtures on 12% denaturing polyacrylamide gels.

In vivo analysis of SMG6 and SMG5 PIN domains. (A–C) S2 cells were transfected with the F-Luc-5BoxB reporter, a plasmid expressing Renilla luciferase, and vectors expressing the λN-protein fusions. Firefly luciferase activity was normalized to that of Renilla and set to 100 in cells expressing the λN-MBP. Mean values±standard deviations from three independent experiments (n=3) are shown. In (C), representative RNA samples corresponding to (B) were analyzed by Northern blot. (D) Decay of the F-Luc-5BoxB mRNA was monitored in cells expressing the different λN-protein fusions. The levels of the F-Luc-5BoxB mRNA normalized to rp49 are plotted against time. mRNA half-lives (t_1/2) calculated from the decay curves are indicated. (E, F) Conserved solvent-exposed residues in the hSMG6-PIN are required for RNA degradation. Residues discussed in the text are shown on the structure and indicated (E). In particular, R1393, R1396, R1402 and W1415 correspond to residues of Drosophila SMG6-PIN (R919, R922, R928 and W942, respectively) tested in the tethering assays in (F). (G) S2 cells were transfected with vectors expressing adh-wt or adh-64 (which carries a PTC at codon 64). A truncated version of adh (adhΔ) served as a transfection control. Vectors expressing dSMG6 wild type, dSMG6 mutant (D881N, D918N) or the corresponding empty vector (control) were included in the transfection mixtures as indicated. Total RNA samples were isolated and analyzed by Northern blot using a probe specific for adh mRNA (not shown). The levels of the adh mRNA reporters were quantitated, normalized to the levels of adhΔ mRNA. These levels were set to 100% for adh-wt in each experimental condition. Mean values±standard deviations of four independent experiments are shown.