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

Tan D, Zhou M, Kiledjian M, Tong L - Nat. Struct. Mol. Biol. (2014)

Bottom Line: 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).

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Columbia University, New York, New York, USA.

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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Structure of the ROQ domain of human Roquin in complex with the constitutive decay element (CDE) of Hmgxb3 mRNA(a). Domain organization of human Roquin. Domains I, II and III of the ROQ domain are colored yellow, cyan and green, respectively. ZF: zinc-finger. (b). Schematic drawing of three RNA molecules used in this study, Hmg19, TNF23, and TNFds. The numbering scheme of the nucleotides is also shown. (c). Two views of the structure of the ROQ domain of human Roquin in complex with Hmg19. The three sub-domains of the ROQ domain are colored in yellow, cyan and green, respectively, and the RNA in orange. The side chain of Met199 (site of the sanroque mutation) is shown in red for carbon atoms. (d). Overlay of the structures of domain III of ROQ domain (green) in complex with Hmg19 (orange) and domain II of ROQ domain (cyan) with TNF23 duplex (yellow). All the structure figures were produced with PyMOL (www.pymol.org).
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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(a). Domain organization of human Roquin. Domains I, II and III of the ROQ domain are colored yellow, cyan and green, respectively. ZF: zinc-finger. (b). Schematic drawing of three RNA molecules used in this study, Hmg19, TNF23, and TNFds. The numbering scheme of the nucleotides is also shown. (c). Two views of the structure of the ROQ domain of human Roquin in complex with Hmg19. The three sub-domains of the ROQ domain are colored in yellow, cyan and green, respectively, and the RNA in orange. The side chain of Met199 (site of the sanroque mutation) is shown in red for carbon atoms. (d). Overlay of the structures of domain III of ROQ domain (green) in complex with Hmg19 (orange) and domain II of ROQ domain (cyan) with TNF23 duplex (yellow). All the structure figures were produced with PyMOL (www.pymol.org).

Mentions: Human Roquin contains 1133 amino acid residues (125 kD, Fig. 1a). The N-terminal RING finger, ROQ and CCCH-type zinc finger (ZF) domains are well conserved among metazoans (Supplementary Fig. 1). The ROQ domain does not share recognizable sequence conservation with other proteins. It is required for binding the CDE, while the RING and ZF domains are dispensable for this interaction 5. The ROQ domain of Roquin-2 is 88% identical to that of Roquin (Supplementary Fig. 1) and Roquin-2 can complement Roquin’s function in repressing autoimmunity 9,10. The C-terminal segment of Roquin following the ZF is poorly conserved in sequence (Supplementary Fig. 1), but it recruits the deadenylation machinery and is required for CDE-mediated decay 5,6.


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

Tan D, Zhou M, Kiledjian M, Tong L - Nat. Struct. Mol. Biol. (2014)

Structure of the ROQ domain of human Roquin in complex with the constitutive decay element (CDE) of Hmgxb3 mRNA(a). Domain organization of human Roquin. Domains I, II and III of the ROQ domain are colored yellow, cyan and green, respectively. ZF: zinc-finger. (b). Schematic drawing of three RNA molecules used in this study, Hmg19, TNF23, and TNFds. The numbering scheme of the nucleotides is also shown. (c). Two views of the structure of the ROQ domain of human Roquin in complex with Hmg19. The three sub-domains of the ROQ domain are colored in yellow, cyan and green, respectively, and the RNA in orange. The side chain of Met199 (site of the sanroque mutation) is shown in red for carbon atoms. (d). Overlay of the structures of domain III of ROQ domain (green) in complex with Hmg19 (orange) and domain II of ROQ domain (cyan) with TNF23 duplex (yellow). All the structure figures were produced with PyMOL (www.pymol.org).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4125485&req=5

Figure 1: Structure of the ROQ domain of human Roquin in complex with the constitutive decay element (CDE) of Hmgxb3 mRNA(a). Domain organization of human Roquin. Domains I, II and III of the ROQ domain are colored yellow, cyan and green, respectively. ZF: zinc-finger. (b). Schematic drawing of three RNA molecules used in this study, Hmg19, TNF23, and TNFds. The numbering scheme of the nucleotides is also shown. (c). Two views of the structure of the ROQ domain of human Roquin in complex with Hmg19. The three sub-domains of the ROQ domain are colored in yellow, cyan and green, respectively, and the RNA in orange. The side chain of Met199 (site of the sanroque mutation) is shown in red for carbon atoms. (d). Overlay of the structures of domain III of ROQ domain (green) in complex with Hmg19 (orange) and domain II of ROQ domain (cyan) with TNF23 duplex (yellow). All the structure figures were produced with PyMOL (www.pymol.org).
Mentions: Human Roquin contains 1133 amino acid residues (125 kD, Fig. 1a). The N-terminal RING finger, ROQ and CCCH-type zinc finger (ZF) domains are well conserved among metazoans (Supplementary Fig. 1). The ROQ domain does not share recognizable sequence conservation with other proteins. It is required for binding the CDE, while the RING and ZF domains are dispensable for this interaction 5. The ROQ domain of Roquin-2 is 88% identical to that of Roquin (Supplementary Fig. 1) and Roquin-2 can complement Roquin’s function in repressing autoimmunity 9,10. The C-terminal segment of Roquin following the ZF is poorly conserved in sequence (Supplementary Fig. 1), but it recruits the deadenylation machinery and is required for CDE-mediated decay 5,6.

Bottom Line: 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).

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Columbia University, New York, New York, USA.

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.

Show MeSH
Related in: MedlinePlus