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The ROQ domain of Roquin recognizes mRNA constitutive-decay element and double-stranded RNA

Nature Structural & Molecular Biology volume 21, pages 679–685 (2014)Cite this article

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Abstract

A conserved stem-loop motif of the constitutive decay element (CDE) in the 3′ UTR of mRNAs is recognized by the ROQ domain of Roquin, which mediates mRNA degradation. Here we report two crystal structures of the Homo sapiens ROQ domain in complex with CDE RNA. The ROQ domain has an elongated shape with three subdomains. The 19-nt Hmgxb3 CDE is bound as a stem-loop to domain III. The 23-nt TNF RNA is bound as a duplex to a separate site at the interface between domains I and II. Mutagenesis studies confirm that the ROQ domain has two separate RNA-binding sites, one for stem-loop RNA (A site) and the other for double-stranded RNA (B site). Mutation in either site perturbs the Roquin-mediated degradation of HMGXB3 and IL6 mRNAs in human cells, demonstrating the importance of both sites for mRNA decay.

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Figure 1: Structure of the ROQ domain of human Roquin in complex with the constitutive decay element (CDE) of Hmgxb3 mRNA.
Figure 2: Interactions between the Hmg19 CDE and the ROQ domain.
Figure 3: Structure of the ROQ domain of human Roquin in complex with the CDE of TNF mRNA.
Figure 4: Biochemical characterization of the ROQ domain–RNA interactions.
Figure 5: Two separate RNA-binding sites in the ROQ domain.
Figure 6: Functional study of Roquin in human HEK293T cells.

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Acknowledgements

We thank X. Jiao (Rutgers University) for constructing the pTK-IREShyg and pIREShyg-Flag plasmids and N. Whalen, R. Jackimowicz and H. Robinson for access to the X29A beamline at the National Synchrotron Light Source. The in-house instrument for X-ray diffraction screening was purchased with a US National Institutes of Health (NIH) grant S10OD012018 (LT). This research is supported by NIH grants GM077175 (L.T.) and GM067005 (M.K.).

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Authors and Affiliations

  1. Department of Biological Sciences, Columbia University, New York, New York, USA

    Dazhi Tan & Liang Tong

  2. Department of Cell Biology and Neuroscience, Rutgers University, Piscataway, New Jersey, USA

    Mi Zhou & Megerditch Kiledjian

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  1. Dazhi Tan
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Contributions

D.T. carried out protein expression, purification and crystallization, X-ray diffraction data collection and processing, structure determination and refinement, mutagenesis and EMSA experiments. M.Z. carried out the mRNA stability experiments. L.T. and M.K. supervised the experiments, analyzed the data and wrote the manuscript. All authors commented on the manuscript.

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Correspondence to Liang Tong.

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Integrated supplementary information

Supplementary Figure 1 Sequence alignment of Roquin proteins.

Sequence alignment of human (Hs), rat (Rn) and Danio rerio (Dr) Roquin (R1) and human Roquin-2 (R2). The secondary structure elements in the structure of the ROQ domain of human Roquin are indicated. Blue and red asterisks denote residues that interact with the Hmg19 and TNF23 RNA, respectively. Magenta triangles indicate residues predicted to coordinate the Zn atom in the zinc-finger motif.

Supplementary Figure 2 Structural similarity between domains II and III of the ROQ domain with winged-helix (WH) domains.

(a). Overlay of the structures of domain III of ROQ domain (in green) and the WH-B domain of cullin 1 (in gray). (b). Overlay of the structures of domain III of ROQ domain (green) and the WH motif of elongation factor SelB in complex with SECIS RNA (gray). (c). Overlay of the structures of domain II of ROQ domain (cyan) and the WH motif of ADAR1 in complex with Z DNA (gray). Produced with the program PyMOL (www.pymol.org).

Supplementary Figure 3 Sequence conservation of the CDE stem-loop among selected mRNAs.

(a). Alignment of CDE sequences in the 3' UTR of TNF α mRNA from various species, and the 3' UTR of other CDE-containing mRNAs. There may be a bulge in the 3' arm of the human IL-6 mRNA. (b). A schematic of an RNA that could bind simultaneously to both A and B sites of the ROQ domain.

Supplementary Figure 4 Two views of the electrostatic surface of the ROQ domain.

(Top) The Hmg19 RNA is bound to a mostly electropositive area in the surface of the WH motif in domain III. (Bottom) The TNF23 RNA is bound to a groove between domains I and II, with a mostly electropositive area in domain I. The two views on the right are nearly the same, and the two views on the left are related by 180° rotation around the vertical axis.

Supplementary Figure 5 TNF23 RNA is bound as a duplex in the crystal.

(Top Left) Two views of the composite omit Fo–Fc electron density map at 1.9 Å resolution for the double-stranded TNF23 RNA, contoured at 2.5σ. The red arrow points to the break in one chain, where nucleotide U10 is disordered. (Top Right) Schematic representation of the structure of the TNF23 duplex. (Bottom) Two views of the structure of TNF23 duplex in complex with two ROQ domains (in color and gray, respectively). There are no contacts with the nucleotides in the middle of the duplex, which correspond to the loop in the stem-loop structure. The red arrows point to the disordered U10 residue in one of the TNF23 molecules.

Supplementary Figure 6 Detailed interactions between the ROQ domain and the TNF23 RNA.

(a). Interactions of Arg131 with the G17 base in the ROQ-TNF23 complex (gray) where U1 and A–1 are disordered. The G17 base from the other complex is also shown (in orange). The red arrow indicates the change in the position of this base in the two complexes. (b). Interactions of Arg131 with the 3' end of the duplex and the 3' flanking sequence in the ROQ-TNF23 complex where U1 is flipped out. (c). Overlay of the structures of the two ROQ-TNF23 complexes in the crystal. One complex is in color, the other in gray. The overlay is based on the protein Cα atoms only. (d). Differences in the binding mode and conformation of the two TNF23 RNA molecules. (e). The local environment of Met199 in the structure of the ROQ domain. (f). The side chain of Met199 is partly exposed to the solvent. A partially transparent surface of domain III is shown.

Supplementary Figure 7 Uncropped images for Figure 4b.

The lanes that are shown in Fig. 4b (middle panel) are highlighted with the red boxes

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Supplementary Figures 1–7, Supplementary Table 1 and Supplementary Notes 1–4 (PDF 12339 kb)

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Tan, D., Zhou, M., Kiledjian, M. et al. The ROQ domain of Roquin recognizes mRNA constitutive-decay element and double-stranded RNA. Nat Struct Mol Biol 21, 679–685 (2014). https://doi.org/10.1038/nsmb.2857

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