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. 2015 Oct 22:5:15660.
doi: 10.1038/srep15660.

New Insights into the RNA-Binding and E3 Ubiquitin Ligase Activities of Roquins

Qi Zhang  1 , Lixin Fan  2 , Feng Hou  1 , Aiping Dong  1 , Yun-Xing Wang  3 , Yufeng Tong  1   4
Affiliations

New Insights into the RNA-Binding and E3 Ubiquitin Ligase Activities of Roquins

Qi Zhang et al. Sci Rep. .

Abstract

Roquins are a family of highly conserved RNA-binding proteins that also contain a RING-type E3 ubiquitin ligase domain. They repress constitutive decay elements containing mRNAs and play a critical role in RNA homeostasis and immunological self-tolerance. Here we present the crystal structures of the RNA-binding region of Roquin paralog RC3H2 in both apo- and RNA-bound forms. The RNA-binding region has a bipartite architecture composed of ROQ and HEPN domains, and can bind to stem-loop and double-stranded RNAs simultaneously. The two domains undergo a large orientation change to accommodate RNA duplex binding. We profiled E2 ubiquitin-conjugating enzymes that pair with Roquins and found that RC3H1 and RC3H2 interact with two sets of overlapping but not identical E2 enzymes to drive the assembly of polyubiquitin chains of different linkages. Crystal structures, small-angle X-ray scattering, and E2 profiling revealed that while the two paralogs are highly homologous, RC3H2 and RC3H1 are different in their structures and functions. We also demonstrated that RNA duplex binding to RC3H2 cross-talks with its E3 ubiquitin ligase function using an in vitro auto-ubiquitination assay.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Characterization of the apo-form RC3H2 structure and comparison with RC3H1.
(A) Ribbon diagram of the structure of the RC3H2 RNA-binding region. The ROQ and HEPN domains are coloured in green and cyan respectively. (B) Domain organization of full-length human RC3H1 and RC3H2. (C) Superposition of apo-RC3H2 (PDB:4Z30) and apo-RC3H1 structures (PDB:4TXA). The two structures are aligned in reference to the HEPN domain. The colour scheme for RC3H2 is the same as that in panel (A); RC3H1 is shown in yellow and zinc ions are shown as grey spheres. (D) SAXS envelope models of RC3H1 (a.a. 1–445, cyan) and RC3H2 (a.a. 1–442, red). For model fitting, the HEPN+ROQ domains of RC3H1 (PDB:4TXA) are replaced with the apo-structure of RC3H2, while keeping the orientation between the HEPN(cyan) and RING (yellow) domains unchanged. The ZnF domain (magenta) is built from PDB:1RGO. The structures were fitted to the SAXS envelope of RC3H2.
Figure 2
Figure 2. Interaction of RC3H2 with RNAs.
(A) Ribbon diagram of the complex structure of RC3H2 and 15-nt Ier3 RNA in an ASU. (B) Schematic drawing of the RNA molecules shown in the structure. The numbering scheme of the nucleotides is also shown. (C) Interaction details of the stem-loop form Ier3 RNA and the ROQ domain. A water molecule bridging the pyrimidine base of U9 and the guanidinium group of Arg248 is shown as a blue sphere.
Figure 3
Figure 3. Interaction details of RC3H2 with double-stranded RNA.
(A) Schematic drawing of RC3H2-Ier3 RNA duplex interactions. W stands for a water molecule. The suffix A or B in the amino acid annotation stands for the protein chains in an ASU. (B) Close-up view of the interactions between the side chain of Arg128 and the purine base of nucleotide G8. (C) Comparison of the different dsRNA-binding modes of RC3H2/Ier3 and RC3H1/Tnf23. Cartoon representation prepared by Q.Z.
Figure 4
Figure 4. Characterization of the interaction of RC3H2 with RNAs in solution.
(A) Size-exclusion chromatography of the RNA-binding region of RC3H2 in the absence and presence of Ier3 and Tnf23 RNAs. (B) Isothermal titration calorimetry measurement of the interactions between RC3H2 RNA-binding region with 15-nt Ier3 (left) and 23-nt Tnf RNAs (right). Upper panel, thermogram; lower panel, data fitted to one-binding site model.
Figure 5
Figure 5. E2 ubiquitin conjugating enzyme profiling for Roquin E3 ubiquitin ligases.
Ubiquitin (green) and His-tag (red) were visualized by immunoblotting after an auto-ubiquitination reaction catalyzed by RC3H2 (A), RC3H1 (B), or without E3 (C). The His-tag of the RC3H2 protein was removed, while that of RC3H1 was intact. All the E2 enzymes were His-tagged except UBE2K, which was GST-tagged. The E1 enzyme was not tagged. A set of control experiments for all the reaction systems without adding ubiquitin is shown in Fig. S9. Free ubiquitin, diubiquitin, and polyubiquitin chain bands are indicated by Ubfree, di-Ub, and Ub(n), respectively. Free ubiquitin bands in panel (A) were mostly ran out of the gel. The free ubiquitin bands overlap with signals from the SDS-PAGE loading dye, so the intensity is not a reliable estimate of the amount. See main text for further discussion.
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