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RNA recognition by Roquin in posttranscriptional gene regulation

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Posttranscriptional regulation of gene expression plays a central role in the initiation of innate and adaptive immune responses. This is exemplified by the protein Roquin, which has attracted great interest during the past decade owing to its ability to prevent autoimmunity. Roquin controls T‐cell activation and T helper cell differentiation by limiting the induced expression of costimulatory receptors on the surface of T cells. It does so by recognizing cis regulatory RNA‐hairpin elements in the 3′ UTR of target transcripts via its ROQ domain—a novel RNA‐binding fold—and triggering their degradation through recruitment of factors that mediate deadenylation and decapping. Recent structural studies have revealed molecular details of the recognition of RNA hairpin structures by the ROQ domain. Surprisingly, it was found that Roquin mainly relies on shape‐specific recognition of the RNA. This observation implies that a much broader range of RNA motifs could interact with the protein, but it also complicates systematic searches for novel mRNA targets of Roquin. Thus, large‐scale approaches, such as crosslinking and immunoprecipitation or systematic evolution of ligands by exponential enrichment experiments coupled with next‐generation sequencing, will be required to identify the complete spectrum of its target RNAs. Together with structural analyses of their binding modes, this will enable us to unravel the intricate complexity of 3′ UTR regulation by Roquin and other trans‐acting factors. Here, we review our current understanding of Roquin–RNA interactions and their role for Roquin function. WIREs RNA 2016, 7:455–469. doi: 10.1002/wrna.1333 This article is categorized under: RNA Interactions with Proteins and Other Molecules > Protein–RNA Recognition RNA Interactions with Proteins and Other Molecules > RNA–Protein Complexes RNA Turnover and Surveillance > Regulation of RNA Stability
The ROQ domain contains two RNA‐binding sites. (a) Schematic representation of the extended Roquin ROQ domain (with subdomains color coded as in Figure ) in complex with either a CDE‐type stem‐loop RNA (pink) at the A‐site or dsRNA (orange) at the B‐site. While canonical A‐site binding involves the core WH motif, dsRNA is bound at a distinct site remote from the A‐site. (b) Structure of the CDE stem‐loop bound to the A‐site in the ROQ domain (PDB 4QI2,). (c) Structure of duplex RNA bound at the B‐site (PDB 4QIK). In this structure (PDB 4QIK), the dimerization of ROQ observed in the crystal lattice may be a consequence of, or be facilitated by binding to the dsRNA.
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The Roquin ROQ domain. Overview of proteolytic fragments harboring the N‐terminal RNA‐binding region of Roquin‐1. (a) The ROQ domain identified by Schlundt et al., with additional flanking helical motifs reported by Tan et al. (b) and by Srivastava et al. (c). The core WH motif fold and the helical extension/domain II are shown in dark and light blue, respectively. The additional helical bundle representing domain I/HEPNN/C and the separate helical extension (black) reported by Srivastava et al. are depicted in gold and black, respectively. The white sections in C indicate regions for which no structural information could be obtained due to lack of electron density. The double‐headed arrow at the top delineates the extent of the core ROQ domain as defined by Schlundt et al. Cartoon representations of the three‐dimensional structures of these fragments are also shown. (d) The ROQ domain (PDB 4QI0) as defined in (a). (e) The region shown in (b) (PDB 4QIK,). (f) The region shown in (c) (PDB 4TXA,). This last structure model is also shown in a rotated view that reveals the RING domain. Colors are as in (a)–(c). Note that the structure shown in (e) depicts the conformation of the ROQ domain when bound to RNA, but for clarity only the protein part is illustrated.
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Roquin function and domain organization. (a) Roquin acts as an mRNA repressor. The scheme shows that the Roquin protein binds to a cis regulatory element in the 3′ UTR of a target mRNA. Typical target mRNAs encode costimulatory T cell receptors, cytokines, and transcription factors. Subsequent to mRNA binding Roquin recruits the deadenylation machinery (CCR4–Not–Caf) and stimulates decapping of the mRNA. CDS is coding sequence. (b) Roquin domain organization. The murine Roquin‐1 protein consists of 1130 amino acids and comprises an N‐terminal RING domain, a ROQ domain (blue: ROQ domain, ocher: extended helical domain (HEPN), see Figure ) and a zinc‐finger (ZnF) domain. The C‐terminal half of Roquin harbors a proline‐rich sequence (PRS) and a short coiled‐coil stretch.
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Summary of RNA binding by Roquin and its regulation. (a) Roquin domains and their involvement in RNA‐ and protein binding. The extended ROQ domain can bind to stem‐loop RNA at the A‐site and double‐stranded RNA at the B‐site, colors as in Figures and (CDS: coding sequence, Ub: ubiquitin). Note that only the Roquin N‐terminal region is depicted schematically. The C‐terminal region (dashed line) is involved in subsequent downstream events that are not indicated here. A role for the Roquin RING domain for E3‐ligase activity is still under investigation; possible targets are unknown with the exception of the data published by Ichijo and colleagues. The zinc‐finger probably supports target recognition by binding to AU‐rich elements in the target mRNA. (b) The efficacy of mRNA repression by the Roquin protein depends on its concentration in the cytoplasm (regulated by the Malt1 protease), the affinity and number of cis RNA elements in the mRNAs available for Roquin binding, and possibly on simultaneous binding of dsRNA regions. The degree of suppression of mRNA translation ranges from ‘not measurable’ (light blue transcript, arrow indicates translation) via ‘medium’ (blue, partial suppression) to ‘strong’ (lilac, complete blockage), where multiple elements are bound and a high concentration of Roquin facilitates nonspecific interaction with dsRNA to guide Roquin to regulatory response elements or possible engagement of nonconsensus response elements.
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The ROQ domain of Roquin recognizes a relaxed stem‐loop RNA consensus. (a) Wild‐type Tnf CDE stem‐loop (23 nt). (b) Structure of the Tnf CDE stem‐loop RNA when bound to the ROQ domain (PDB 4QI2; note that the protein is not shown). (c) The table shows variants of the 23‐mer CDE in (a) that were tested in in vitro affinity assays by Schlundt et al. and Leppek et al. The relative change in affinity compared to the WT is indicated by the color code as follows: no change (green), less than fivefold reduction (orange), complete loss of binding (red). The nucleotide variations are highlighted in yellow or deleted from the scheme. Binding of single‐stranded RNA was tested by NMR titrations. (d) Relaxed CDE consensus based on panel (c). (e) Adapted CDE consensus based on mRNA decay assays in references and suggested by Codutti et al. (f) A general suggestion for a CDE consensus based on all available data (i.e., those reflected in (d) and (e), recent findings with U‐rich hexa‐loops, and our own data (Janowski et al., under revision).
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Details of Roquin–RNA recognition at the A‐ and B‐sites. (a) Recognition of the Tnf CDE motif by the ROQ domain of Roquin (PDB 4QI2). The ROQ domain interacts with the 5′‐half of the CDE stem of the Tnf mRNA by nonsequence‐specific contacts to the RNA backbone. Key interactions are annotated. (b–d) Close‐up views of the recognition of the Py–Pu–Py tri‐loop of the Tnf CDE (U11, G12, and U13). (e) Recognition of duplex Tnf dsRNA at the B‐site interface by the extended ROQ domain involves mainly nonsequence‐specific contacts with the RNA backbone (PDB 4QIK). (f) A different view of the Roquin–dsRNA complex in the B‐site from that shown in (d). Colors in all panels are the same as in Figure .
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RNA Turnover and Surveillance > Regulation of RNA Stability
RNA Interactions with Proteins and Other Molecules > RNA–Protein Complexes
RNA Interactions with Proteins and Other Molecules > Protein–RNA Recognition

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