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Structural basis for RNA recognition in roquin-mediated post-transcriptional gene regulation


Roquin function in T cells is essential for the prevention of autoimmune disease. Roquin interacts with the 3′ untranslated regions (UTRs) of co-stimulatory receptors and controls T-cell activation and differentiation. Here we show that the N-terminal ROQ domain from mouse roquin adopts an extended winged-helix (WH) fold, which is sufficient for binding to the constitutive decay element (CDE) in the Tnf 3′ UTR. The crystal structure of the ROQ domain in complex with a prototypical CDE RNA stem-loop reveals tight recognition of the RNA stem and its triloop. Surprisingly, roquin uses mainly non-sequence-specific contacts to the RNA, thus suggesting a relaxed CDE consensus and implicating a broader spectrum of target mRNAs than previously anticipated. Consistently with this, NMR and binding experiments with CDE-like stem-loops together with cell-based assays confirm roquin-dependent regulation of relaxed CDE consensus motifs in natural 3′ UTRs.

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Figure 1: The ROQ domain is sufficient for CDE RNA binding.
Figure 2: Structure of the roquin-1 ROQ–Tnf CDE RNA complex.
Figure 3: Mutational analysis of the ROQ–Tnf CDE RNA interface.
Figure 4: Functional analysis of roquin-1 ROQ-domain mutations.
Figure 5: Mutational analysis of the Tnf CDE RNA.
Figure 6: Functional analysis of CDE-like RNA variations.

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We acknowledge the use of the X-ray crystallography platform and the protein expression and purification facility of the Helmholtz Zentrum München, the Bavarian NMR Center, and SAXS measurements at the facility of the SFB1035, Technische Universität München. We thank S. Hauck (Helmholtz Zentrum München) and J. Winter (Technische Universität München) for MS analysis and A. Berns (The Netherlands Cancer Institute) for providing the Cre-ERT2 mouse line. G.A.H. acknowledges the Boehringer Ingelheim Fonds for a PhD fellowship. This work was supported by the Deutsche Forschungsgemeinschaft through grants SCHL2062/1-1 (to A.S.), SFB1035 and GRK1721 (to M.S.), NI-1110/4-1 (to D.N.), SFB646 and FOR855 (to D.N.) and SFB1054 TP-A03 (to V.H.) and by the European Commission through a European Research Council grant to V.H.

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Authors and Affiliations



A.S. and A.G. carried out cloning, protein expression and static light scattering. A.S. performed and analyzed NMR experiments. R.J. performed crystallization and structure determination. G.A.H. carried out EMSA assays and functional experiments. A.S. and R.S. recorded and analyzed SAXS data. A.S., G.A.H., R.J., V.H., D.N. and M.S. designed the project and wrote the paper. All authors discussed the results and commented on the manuscript.

Corresponding authors

Correspondence to Vigo Heissmeyer, Dierk Niessing or Michael Sattler.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 The minimum RNA-binding domain of roquin-1.

(a) SDS-PAGE analysis of roquin-1 (64-411) after digestion with protease K as described in the method section. The band indicated by the arrow was used for identification via tryptic digestion and complete mass determination and revealed a fragment of approx. 17.8 kDa between amino acids 150 and 330. (b) Superposition of NMR HSQC spectra of 15N-labeled roquin-1 (64-411) after treatment with protease K (green) or trypsin (red). Minor differences in peak position and numbers are due to different overhangs produced by the proteases. (c) HSQC overlay of roquin-1 (64-411) after treatment with protease K (green) and a recombinant roquin-1 (147-326, ROQ) protein (black). (d) Sequence alignment of roquin-1 ROQ (147-326) showing isotype comparison and primary sequences from organisms as indicated. Secondary structure elements as subsequently determined from the crystal structure (Fig. 1f) are shown on top. Asterisks indicate residues that are involved in RNA contacts based on the crystal structure (Fig. 2a, b) or show significant chemical shift perturbations upon binding to Tnf CDE RNA (Fig. 3a, c) in NMR titrations. Residues used for mutational analyses and the corresponding amino acid substitutions are indicated by capital letters. The K314A mutation was chosen as negative control.

Supplementary Figure 2 NMR analysis of roquin-1 ROQ and ROQ–CDE RNA.

(a) Secondary 13C chemical shifts Δδ(13Cα-13Cβ), (b) heteronuclear {1H}-15N NOE ratios, (c,d) 15N R1 and R2 relaxation rates and (e) τc values derived from the R2/R1 ratio of roquin-1 ROQ alone. (f) Secondary 13Cα chemical shifts and (g) heteronuclear {1H}-15N NOE ratios of the ROQ domain in complex with Tnf CDE RNA. Relaxation data for residues 171-326 were used to estimate the tumbling correlation time based on a trimmed mean R2/R1 ratio as described in the methods section. Error bars for the heteronuclear NOE data are obtained by error propagation of peak height uncertainties based on average noise levels in the NMR spectra. Relaxation rate errors represent fit deviations and are propagated into the calculation of an error for the tumbling correlation times. The secondary structure elements seen in the ROQ crystal structure are shown on top.

Supplementary Figure 3 Structure and biophysical characterization of the ROQ domain.

(a) Structure of the ROQ domain (147-326) in the asymmetric unit of the crystals in two different orientations (90° rotation). Chain A is shown in green, chain B (with electron density observed for more residues at its N-terminus) is shown as follows: the α-helices of the winged helix (WH) are shown in cyan except of the recognition helix, which is shown in yellow. The three-stranded β sheet is shown in blue and the remaining part of the structure in gray. (b) Zoomed view of the ROQ domain showing electron density for the recognition helix α4. (c) Roquin-1 oligomerization probed by static light scattering. Combined analytical gel filtration chromatograms displaying UV absorption and refractive indices (see color code) of roquin-1 ROQ (top panel, theoretical MW: 20.5 kDa, 7 mg/ml) and the roquin-1 N terminal domain (bottom panel, theoretical molecular weight (MW): 49 kDa, 3.7 mg/ml) with MWs calculated from the Omnisec software using the indicated corresponding peak. Insets show the main peak of interest (indicated by an arrow) with their refractive indices and molecular weight curves plotted as binary logarithm (log2MW), respectively. Samples were run on a SD200 semi-analytical column of 23 ml volume. (d) Superposition of the roquin-1 WH domain (red) with the most similar WH domains identified by structural similarity searches: cell division control protein 53 (gray; PDB 3O6B5; r.m.s.d 1.8 Å) and cullin-1 (cyan; PDB code: 3TDU6; r.m.s.d 2.2 Å). (e) The ROQ domain exhibits conformational variability in the β3-W1-β4 region. Chain A and chain B of the apo-structure is shown in blue and in red, respectively. The ROQ domain from the ROQ-RNA complex is shown in green. The only part of the structures showing considerable conformational differences is the β3-W1-β4 region, which is consistent with the flexibility of this region seen by NMR. In the RNA bound state, this β3-W1-β4 region adopts a more closed and less variable conformation. (f) The roquin-1 ROQ domain (red) is a novel domain fold. Using the SSM server, the transcription antitermination complex from E. coli (PDB code 3D3C7, cyan) with an r.m.s.d. of 3.8 Å and the Q-score of 0.18 was identified as the most similar structure.

Supplementary Figure 4 Structural analysis of the ROQ–Tnf CDE RNA complex.

(a) Electron density map for the Tnf CDE RNA base U4 in the structure of the ROQ-RNA complex shown in Fig. 2a. (b) Electron density map for the Tnf CDE RNA base U11. (c) Tnf CDE RNA hairpin structure when bound to ROQ domain. The ROQ domain is not shown. (d) Structure of the Tnf CDE RNA-ROQ domain complex. The ROQ domain is shown as surface representation colored by electrostatic potential. (e-j) Structure similarity search of the ROQ-RNA complex. All structures are shown with the same orientation for the recognition helix (magenta). (e) Roquin ROQ (197-273) (PDB: ZZZ). (f) Human Z alpha domain of the RNA-editing enzyme ADAR1 (PDB: 2GXB8). (g) and (h) Elongation factor SelB from M. thermoacetica (PDB: 2PLY9) domain WH4 and WH3, respectively. (i) Elongation factor SelB from M. thermoacetica (PDB: 1WSU10), only contacts with the symmetry related RNA molecule shown. (j) Superposition of the crystal structure of the ROQ-Tnf CDE RNA complex and the shape determined from SAXS data of a 2 mg/ml sample of ROQ with 1.1x Tnf CDE RNA. (k) Distance distribution plots of roquin-1 ROQ alone at different concentrations and in complex with Tnf CDE RNA at concentrations as indicated. A summary of calculated parameters from individual samples is given in Supplementary Table 1. (l) Plot of the average particle molecular weight in dependence of concentration as determined with SAXS in (k) and Supplementary Table 1. The MW of apo ROQ is 20.5 kDa and the complex MW is 27 kDa.

Supplementary Figure 5 EMSA and NMR analysis of the ROQ–Tnf CDE RNA interaction.

(a,b) Induced fit of Tnf CDE RNA upon binding of the wild-type ROQ domain. 2D imino proton NOESY spectra of the CDE free (a) and in complex with ROQ (b). The large up-field shift for the NMR signal of the G14 imino proton in the bound RNA is consistent with ring current shift induced by stacking with the closing C-G base pair. On top, the corresponding region of a 1D imino proton spectrum is shown to indicate the number of imino signals before and after ROQ binding. Signals marked with an asterisk are caused by free RNA or protein. (c) EMSA experiments for ROQ mutants with Tnf CDE RNA. Equilibrium dissociation constants determined from quantitative analysis are shown in Fig. 3f and Supplementary Table 2.

Supplementary Figure 6 Functional analysis of roquin-1 ROQ domain mutations by FACS analysis as quantified in Figure 4.

(a) Co-expression of ICOS and roquin-1 (assessed by ICOS and Thy1.1 surface expression) in roquin-1/2-deficient MEFs. The cells were transduced with a retrovirus encoding ICOS full-length mRNA and superinfected with the indicated retroviruses containing wildtype (WT) roquin-1 or roquin-1 mutants linked to an internal ribosomal entry site and the Thy1.1 gene. In flow cytometric analyses cells with and without ectopic roquin-1 expression were defined in the right and left rectangles, respectively. (b) The same assay as in (a) using Ox40 full-length mRNA as reporter and Ox40 surface expression as read-out. (c) The same as in (a) using 260 nucleotides containing the prototypic CDE from the Tnf 3' UTR as artificial 3' UTR fused to the ICOS coding region.

Supplementary Figure 7 NMR analysis of CDE variants and CDE-like RNAs by 2D imino proton NOESY spectra of RNAs to identify base-pairing.

The respective substitutions as compared to the canonical Tnf CDE RNA are highlighted by color code as in Fig. 5. (a) Tnf CDE stem mutant 3. (b) Shown is the Tnf CDE extended mutant. (c,d) The two naturally occurring CDE-like variants from Ox40 and human ICOS mRNAs. All deviations from Tnf CDE are labelled. Only base pairs observed by NMR are indicated as red bonds for the base pairs in the schematic RNA stem loops.

Supplementary Figure 8 Comparison of NMR chemical-shift perturbations (CSPs) for different CDE-like RNAs.

(a) Superposition of 1H,15N HSQC spectra of roquin-1 ROQ (black) free, when bound to Tnf CDE RNA (red) and (left) bound to the CDE-derivative CDE EXT with an extended stem (orange) or (right) bound to the Ox40 CDE (cyan). (b) CSPs (Δδ) of ROQ upon binding to Tnf CDE EXT (top) or Ox40 CDE (bottom). Gray bars indicate residues that are unassigned in one of the spectra, usually the free protein. Gaps derive from prolines or completely unassigned peaks. (c) Differential CSPs for ROQ binding to Tnf CDE and Tnf CDE EXT (top) or Ox40 CDE (bottom). (d) Mapping of significant chemical shift perturbations of amides from (c) (as indicated by color coding). Amides with strong CSP differences are shown as spheres

Supplementary Figure 9 Full-size scans of western blot membranes shown in main figures.

(a) Uncropped original image of immunoblot analysis as shown in Fig. 4a. The left blot shows detection of roquin-1 (125 kDa) and the right one was probed for tubulin (55 kDa) as control with the same order of samples from left to right. The right outer lane was loaded with lysate from non-infected cells (not denoted). Numbers represent molecular weight standards given in kDa. (b) Immunoblotting analysis of tamoxifen treatment of Cre Ert2; CAG-CARstop-fl; Rc3h1-2fl/fl MEFs that results in loss of roquin-1 and - 2. Uncropped original image of immunoblot analysis as shown in Fig. 6c. The left blot shows detection of roquin-1 and -2 (125 and 131 kDa, respectively) in dependence of tamoxifen treatment and the right one was probed for tubulin (55 kDa) as control with the same order of samples from left to right. Numbers represent molecular weight standards given in kDa.

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Schlundt, A., Heinz, G., Janowski, R. et al. Structural basis for RNA recognition in roquin-mediated post-transcriptional gene regulation. Nat Struct Mol Biol 21, 671–678 (2014).

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