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Acta Crystallographica Section F: Structural Biology Communications logoLink to Acta Crystallographica Section F: Structural Biology Communications
. 2016 Apr 22;72(Pt 5):397–402. doi: 10.1107/S2053230X16006129

RRM domain of human RBM7: purification, crystallization and structure determination

Nicholas Sofos a, Mikael B L Winkler a, Ditlev E Brodersen a,*
PMCID: PMC4854568  PMID: 27139832

The X-ray crystal structure of the RRM domain of human RBM7 reveals a pentameric assembly.

Keywords: RNA degradation, human, NEXT complex, RRM domain, X-ray crystallography

Abstract

RNA decay is an important process that is essential for controlling the abundance, quality and maturation of transcripts. In eukaryotes, RNA decay in the 3′–5′ direction is carried out by the exosome, an RNA-degradation machine that is conserved from yeast to humans. A range of cofactors stimulate the enzymatic activity of the exosome and serve as adapters for the many RNA substrates. In human cells, the exosome associates with the heterotrimeric nuclear exosome targeting (NEXT) complex consisting of the DExH-box helicase hMTR4, the zinc-finger protein hZCCHC8 and the RRM-type protein hRBM7. Here, the 2.5 Å resolution crystal structure of the RRM domain of human RBM7 is reported. Molecular replacement using a previously determined solution structure of RBM7 was unsuccessful. Instead, RBM8 and CBP20 RRM-domain crystal structures were used to successfully determine the RBM7 structure by molecular replacement. The structure reveals a ring-shaped pentameric assembly, which is most likely a consequence of crystal packing.

1. Introduction  

RNA surveillance, maturation and decay in eukaryotic cells depend to a great extent on the 3′–5′ exonucleolytic and endonucleolytic activities of the RNA exosome complex (Chlebowski et al., 2013 ▸; Schneider & Tollervey, 2013 ▸). Associated proteins regulate exosome activity and the recruitment of substrates, with the best characterized cofactors being the nuclear Trf4p/5p–Air1p/2p–Mtr4p polyadenylation (TRAMP) complex and the cytoplasmic Ski2p–Ski3p–Ski8p (SKI) complex in Saccharomyces cerevisiae (LaCava et al., 2005 ▸; Wyers et al., 2005 ▸; Vaňáčová et al., 2005 ▸; Anderson & Parker, 1998 ▸; Brown et al., 2000 ▸). In human cells, the nuclear RNA exosome is directed to many of its nucleoplasmic substrates by the nuclear exosome targeting (NEXT) complex, consisting of the DExH-box helicase and yeast Mtr4p homologue hMTR4/SKIV2L2, the RRM-protein RBM7 and the zinc-finger protein ZCCHC8 (Lubas et al., 2011 ▸, 2015 ▸; Andersen et al., 2013 ▸).

Accumulating evidence suggests a role for the NEXT complex in the surveillance of RNA polymerase II transcripts, including pre-mRNAs, PROMPTs, sn(o)RNAs, enhancer RNAs and replication-dependent histone RNAs (Andersen et al., 2013 ▸; Hrossova et al., 2015 ▸; Lubas et al., 2011 ▸, 2015 ▸). RBM7 has been shown to be involved in targeting the NEXT complex to U-rich stretches in RNA, requiring a minimum of four pyrimidine nucleotides for efficient binding (Lubas et al., 2015 ▸; Hrossova et al., 2015 ▸). A recent NMR structure showed that the N-terminal domain of human RBM7 adopts a classical RNA-recognition motif (RRM) α/β-sandwich fold with a β1–α1–β2–β3–α2–β4 topology, which is a putative RNA-binding domain of about 90 amino-acid residues (Hrossova et al., 2015 ▸; Maris et al., 2005 ▸). The RBM7 RRM domain also contains the RNP1 [(RK)-G-(FY)-(GA)-(FY)-(ILV)-X-(FY)] and RNP2 [(ILV)-(FY)-(ILV)-X-N-L] signature sequences characteristic of the RRM family. The only deviation is the second residue of the RNP1 sequence of RBM7, which is Q and not G (Dreyfuss et al., 1988 ▸; Hrossova et al., 2015 ▸). RNA-binding studies have further shown that Phe13 and Phe52 of the first and third β-strands, respectively, are important for RNA binding, most likely through stacking interactions with nucleotide bases (Hrossova et al., 2015 ▸; Maris et al., 2005 ▸).

Here, we report the crystallization, structure determination and structural analysis of the crystal structure of the human RBM7 RRM domain determined to 2.5 Å resolution. The structure shows an unusual pentameric assembly, as well as an extended α2-helix and two short, tentative β-strands compared with the corresponding NMR model.

2. Materials and methods  

2.1. Cloning, expression and purification  

The part of the human RBM7 gene encoding residues 1–91 of the intact protein was inserted into the pET28M-6×His-SUMOTag expression vector (Tomecki et al., 2015 ▸). The resulting plasmid was transformed into Escherichia coli Rosetta 2 (DE3) competent cells, which were grown in LB medium supplemented with 34 µg ml−1 chloramphenicol and 50 µg ml−1 kanamycin at 37°C until the OD600 reached 0.6–0.8. Cultures were chilled in ice water before expression at 18°C by the addition of 0.5 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) overnight. The cells were harvested and resuspended in 50 mM HEPES pH 7.0, 0.5 M KCl, 40 mM imidazole, 10 mM MgCl2, 10% glycerol, 3 mM β-mercaptoethanol and protease-inhibitor cocktail tablets (Sigma–Aldrich) and were subsequently lysed by sonication and centrifuged at 30 000g for 30 min to remove cell debris. The supernatant was loaded onto a pre-equilibrated 5 ml Ni–NTA column (Qiagen). The bound protein was washed with 1 M KCl and eluted with 50 mM HEPES pH 7.0, 0.25 M KCl, 0.3 M imidazole, 10%(v/v) glycerol, 3 mM β-mercaptoethanol and subsequently exchanged into 50 mM HEPES pH 7.0, 0.3 M KCl, 10% glycerol, 5 mM β-mercaptoethanol using a HiPrep 26/10 desalting column (GE Healthcare). The His-SUMO tag was released by treatment with Ulp protease (a kind gift from A. Dziembowski, Warsaw University; Mossessova & Lima, 2000 ▸) and subsequently removed by reapplying the sample onto the Ni2+-affinity column. Finally, RBM7 1–91 was purified by gel-filtration chromatography on a Superdex 75 10/300 column (GE Healthcare) equilibrated in 20 mM HEPES pH 7.0, 150 mM KCl, 5 mM β-mercaptoethanol and was concentrated to ∼10 mg ml−1 using a 5000 molecular-weight cutoff spin filter (Sartorius).

2.2. Crystallization, data collection and structure determination  

Human RBM7 1–91 was crystallized at 19°C using a reservoir solution consisting of 0.1 M HEPES pH 7.5, 1.4 M tri­sodium citrate with 0.2 + 0.2 µl sitting-drop vapour-diffusion drops. Rhombic crystals appeared within 4 d and grew to a maximum size of 100–150 µm in two dimensions. The crystals were transferred to a drop of mother liquor supplemented with 10%(v/v) glycerol and immediately flash-cooled in liquid nitro­gen. Diffraction data extending to 2.5 Å resolution were collected on beamline I04 at Diamond Light Source, where 2400 fine-sliced diffraction images were collected with a 0.10° oscillation angle from a single crystal and were auto­processed with xia2 via XDS/XSCALE (Winter et al., 2013 ▸; Kabsch, 2010 ▸). The crystals belonged to space group C121 with five molecules per asymmetric unit. Structure determination was carried out by molecular replacement in Phaser using an ensemble search model consisting of PDB entries 2j0q (Bono et al., 2006 ▸) and 1h2v (Mazza et al., 2002 ▸). The successful MR solution was subjected to one round of automatic model building and refinement in ARP/wARP Classic as implemented in the CCP4 suite (Morris et al., 2003 ▸; Perrakis et al., 2001 ▸; Winn et al., 2011 ▸). Subsequently, the model was manually rebuilt in Coot (Emsley et al., 2010 ▸) and iteratively refined using phenix.refine (Afonine et al., 2012 ▸) and REFMAC (Murshudov et al., 2011 ▸). Data-collection, processing and refinement statistics are shown in Table 1 ▸.

Table 1. Data-collection and refinement statistics for human RBM7 1–91.

Values in parentheses are for the outer resolution shell.

Data collection
 PDB code 5iqq
 Wavelength (Å) 0.9720
 Resolution range (Å) 49.1–2.5 (2.6–2.5)
 Space group C121
 Unit-cell parameters (Å, °) a = 115.71, b = 83.21, c = 60.83, α = 90, β = 119.63, γ = 90
 Observed reflections 78050 (5716)
 Unique reflections 17201 (1266)
  R meas (%) 17.7 (116.5)
 CC1/2 (%) 99.3 (47.7)
 Multiplicity 4.5 (4.5)
 Completeness (%) 96.8 (97.2)
 Mean I/σ(I) 10.6 (1.5)
Refinement
 Reflections used in refinement 16761 (1632)
 Reflections used for R free 848 (98)
  R (%) 21.6 (29.6)
  R free (%) 24.6 (35.7)
 No. of non-H atoms
  Total 3521
  Protein 3267
  Solvent 254
 No. of protein residues 405
 R.m.s.d., bond lengths (Å) 0.004
 R.m.s.d., bond angles (°) 0.70
 Ramachandran favoured (%) 95.0
 Ramachandran allowed (%) 3.0
 Ramachandran outliers (%) 2.0
 Average B factor (Å2)
  Overall 45.9
  Macromolecule 46.1
  Solvent 44.2
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3. Results and discussion  

3.1. Purification of the human RBM7 RRM domain  

Sequence analysis of human RBM7 using GlobPlot and Phyre2 initially predicted the C-terminal region to be largely disordered, and thus residues 1–91 corresponding to the core RBM7 RRM domain were chosen for structural analysis (Kelley et al., 2015 ▸; Linding et al., 2003 ▸). The corresponding DNA sequence was inserted into pET28M-6×His-SUMOTag, giving rise to a protein with dual N-terminal 6×His affinity and SUMO solubility tags (Fig. 1 ▸ a; see §2 for details). Following Ni2+-affinity purification and removal of the dual tag using Ulp protease, monodisperse protein was obtained using gel filtration on a high-resolution Superdex 75 10/300 column (Fig. 1 ▸ b). During gel filtration, the RBM7 RRM domain elutes at 14.2 ml, corresponding to a theoretical molecular mass of approximately 12 kDa. Based on this, we conclude that the isolated RBM7 RRM domain is a monomer in solution.

Figure 1.

Figure 1

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Purification of the human RBM7 RRM domain. (a) A 15% SDS–PAGE stained with Coomassie Brilliant Blue showing the progress of protein purification. His-SUMO-RBM7 1–91 was first isolated by Ni–NTA chromatography before removal of the His-SUMO tag by Ulp protease cleavage and subsequent separation by Ni–NTA chromatography. Lane M contains molecular-mass marker (labelled in kDa). (b) RBM7 1–91 elutes at 14.2 ml on a Superdex 75 10/300 column, corresponding to a molecular weight of ∼12 kDa. The void volume (V 0) and elution volumes of standard proteins are shown above the chromatogram (A 280, blue; A 260, red).

3.2. Crystallization and structure determination  

Rhombic crystals of the RBM7 RRM domain were obtained in 1.4 M citrate pH 7.5 at 19°C. The crystals diffracted to 2.5 Å resolution and belonged to space group C121 with five RBM7 RRM monomers per asymmetric unit. Surprisingly, structure determination by molecular replacement using a recently determined structure of a similar fragment of human RBM7 determined in solution by NMR (PDB entry 2m8h) was unsuccessful (Hrossova et al., 2015 ▸). Instead, a 3D-BLAST structure search was performed using a minimal domain fragment of the NMR structure as a search model (Yang & Tung, 2006 ▸). This search identified RBM8 from the crystal structure of the exon junction complex (PDB entry 2j0q; 28% sequence identity, 49% sequence similarity) and CBP20 derived from the human cap-binding complex (PDB entry 1h2v; 34% sequence identity, 51% sequence similarity) as the closest homologous structures in the PDB (Bono et al., 2006 ▸; Mazza et al., 2002 ▸). Using a search ensemble of these two structures, we were able to obtain a good molecular-replacement solution in Phaser with a Z-score of 11.4 (McCoy et al., 2007 ▸). After one round of automatic rebuilding in ARP/wARP (Morris et al., 2003 ▸; Perrakis et al., 2001 ▸), the structure was completed by iterative refinement in PHENIX and REFMAC (Adams et al., 2010 ▸; Murshudov et al., 2011 ▸), resulting in final R and R free values of 21.6 and 24.6%, respectively (Table 1 ▸). The final model contains five copies of the RBM7 RRM domain including residues 4–86 (chain A), 8–85 (chain B), 3–85 (chain C), 5–85 (chain D) and 6–85 (chain E).

3.3. The human RBM7 RRM domain forms a pentameric assembly  

The five RBM7 molecules per crystallographic asymmetric unit assemble into a pentameric ring structure with fivefold noncrystallographic symmetry (Fig. 2 ▸ a). In the crystal packing, the pentamers are arranged in plates stacked on top of each other, with solvent channels running through the centre of the rings and little room between the individual plates (Fig. 2 ▸ b). Neighbouring molecules only share 1–2 hydrogen bonds and a single hydrophobic interaction between Tyr76 and Pro64 (Fig. 3 ▸). An intersubunit hydrogen-bond interaction between the backbone carbonyl of Lys74 and the side-chain amido NH2 group of Asn68 is consistent throughout the ring assembly, whereas the Tyr76 and Glu60 side chains form hydrogen bonds between two pairs of adjacent molecules (Fig. 3 ▸). Analysis of the contents of the asymmetric unit using PISA revealed that the protein interface areas of the pentamer average around 274 Å2, with a ΔG interaction of around −3.9 kcal mol−1 (Krissinel & Henrick, 2007 ▸). Put together, the low number of interactions and the small size of the interface area strongly indicate that the pentamer is a crystal-packing artefact and thus is consistent with the behaviour of the human RBM7 RRM domain in solution.

Figure 2.

Figure 2

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(a) The asymmetric unit of the RBM7 1–91 crystals contains a pentameric assembly of RRM domains. The model is shown in cartoon representation in various shades of green. (b) Crystal packing of the RBM7 1–91 pentamer structure. Solvent channels run through the centres of the pentamers (top), which form leaflets that are stacked on top of each other in the perpendicular direction (bottom).

Figure 3.

Figure 3

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Final σA-weighted 2DF o − mF c map contoured at 1σ, showing representative electron density at the interface between chains C (dark green) and D (light green). The enlarged view (right) shows details of the intersubunit interactions observed. Subscript letters indicate the chain to which the respective residues belong. Tyr76D and Glu60C form a hydrogen bond, Tyr76D and Pro64C engage in hydrophobic interactions, and the backbone carbonyl O atom of Lys74D forms a hydrogen bond to the side-chain amide of Asn68C.

3.4. Analysis of the RBM7 RRM fold  

The human RBM7 RRM domain adopts a canonical RRM fold in all five molecules in the crystal structure, consisting of two α-helices packed against a four-stranded antiparallel β-sheet (Fig. 4 ▸ a). In addition to the core RRM motif, the structure contains two short, tentative β-strands, which we denote βA and βB (Fig. 4 ▸ c). The latter is an extension of the β4 strand. These were not observed in the NMR structure and are not part of the classical RRM fold (Maris et al., 2005 ▸). There are only minor structural differences between the five molecules in the asymmetric unit (r.m.s.d. on Cα atoms of 0.137 Å), which are primarily located in the N-terminus (residues 4–10) and in the long loop between β2 and β3. Compared with the NMR structure (Hrossova et al., 2015 ▸), helix α2 is longer by four residues (residue 60–63) in the crystal structure, likely owing to the packing interaction between Pro64 and Tyr76 of the adjacent molecule that appears to stabilize the helix despite the presence of a proline. Finally, the loop regions at one end of the β-sheet are slightly further apart in the crystal structure compared with the NMR structure (Fig. 4 ▸ b).

Figure 4.

Figure 4

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(a) Cartoon overview of the human RBM7 RRM domain with secondary-structure elements labelled according to the notation shown in (c). βA and βB (orange) are short, tentative β-strands that are not part of the canonical RRM motif, with the latter being an extension of the β4 strand. (b) Superposition of an ensemble of the 20 lowest energy NMR structures of the human RBM7 RRM domain (light grey) with chain D of the crystal structure (dark green), in the same orientation as in (a). (c) Amino-acid sequence of the N-terminal part of human RBM7 with the extent of the crystal structure shown above and helices (brown) and sheets (grey) annotated as in (a). βA and βB are shown in light grey with dashed outlines. The RNP2 and RNP1 motifs are indicated in the dashed boxes. Gln51, shown with dark blue shading, is a deviation from the RNP1 consensus sequence, which has a glycine at this position.

4. Conclusion  

We have crystallized and determined the crystal structure of the core RRM domain of human RBM7 covering residues 1–91 to 2.5 Å resolution. An ensemble of the homologous RRM-domain structures from CBP20 and RBM8 could successfully be used to determine the structure by molecular replacement. The crystallographic asymmetric unit contains five RRM domains arranged in a pentameric ring structure, but weak intermolecular interactions and a small intersubunit interface, as well as gel-filtration chromatography, strongly suggest that the pentamer is a crystal-packing artefact. The human RBM7 RRM structure is very similar to a recent structure determined in solution by NMR. However, small differences in the loop regions are observed, and α2 is extended in the crystal structure compared with the solution structure. Finally, we observe two short, tentative β-strands, βA and βB, which are not part of the classical RRM fold.

Supplementary Material

PDB reference: human RBM7 RRM domain, 5iqq

Acknowledgments

The authors would like to thank Andrzej Dziembowski and Anna Łabno for providing a full-length clone for human RBM7 in pET28M-6×His-SUMOTag, which was used to construct the RBM7 1–91 fragment, and Thomas Boesen for collecting the diffraction data, as well as the beamline staff at Diamond Light Source beamline I04 (proposal/visit No. mx9317-18). This work was funded by the Danish National Research Foundation’s Centre for mRNP Biogenesis and Metabolism (grant No. DNRF58) and a Novo Nordisk Foundation grant (NNF15OC0017384) to DEB. The authors declare no competing financial interests.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

PDB reference: human RBM7 RRM domain, 5iqq

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