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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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Two separate RNA binding sites in the ROQ domain(a). Overlay of the structures of the Hmg19 complex (in color) and TNF23 complex (gray) of the ROQ domain, based on domains II and III. A 30° change in the orientation of domain I is indicated with the red arrow. (b). EMSA results showing the formation of the ROQ-TNFds-Hmg19 ternary complex. Wild-type ROQ domain was pre-incubated with labeled TNFds, and then with increasing concentrations of unlabeled Hmg19. The NaCl concentration was 50 mM. (c). EMSA results with mutations in the A site. The mutations blocked the formation of the ternary complex. (d). A model showing possible linkages between the RNA molecules in the B site and A site. A single-stranded RNA (gray, 7 nts) was modeled to connect the 3′ end of the RNA in the B site to the 5′ end of the stem-loop RNA in the A site (~30 Å distance). A linker between the 3′ end of the RNA in the A site to the 5′ end of the RNA in the B site would need to span ~60 Å (gray dots).
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Figure 5: Two separate RNA binding sites in the ROQ domain(a). Overlay of the structures of the Hmg19 complex (in color) and TNF23 complex (gray) of the ROQ domain, based on domains II and III. A 30° change in the orientation of domain I is indicated with the red arrow. (b). EMSA results showing the formation of the ROQ-TNFds-Hmg19 ternary complex. Wild-type ROQ domain was pre-incubated with labeled TNFds, and then with increasing concentrations of unlabeled Hmg19. The NaCl concentration was 50 mM. (c). EMSA results with mutations in the A site. The mutations blocked the formation of the ternary complex. (d). A model showing possible linkages between the RNA molecules in the B site and A site. A single-stranded RNA (gray, 7 nts) was modeled to connect the 3′ end of the RNA in the B site to the 5′ end of the stem-loop RNA in the A site (~30 Å distance). A linker between the 3′ end of the RNA in the A site to the 5′ end of the RNA in the B site would need to span ~60 Å (gray dots).

Mentions: The structures of the Hmg19 and TNF23 complexes demonstrate that the ROQ domain has two separate RNA binding sites, with no overlaps between them (Fig. 5a). The binding mode of Hmg19 RNA in the A site clearly indicates that a dsRNA cannot be accommodated there, as the loop makes intimate contacts with the ROQ domain (Fig. 2c). Our mutagenesis and EMSA results confirm that the A site in domain III is for binding stem-loop RNA, while the B site at the interface of domains I and II is for binding dsRNA.


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)

Two separate RNA binding sites in the ROQ domain(a). Overlay of the structures of the Hmg19 complex (in color) and TNF23 complex (gray) of the ROQ domain, based on domains II and III. A 30° change in the orientation of domain I is indicated with the red arrow. (b). EMSA results showing the formation of the ROQ-TNFds-Hmg19 ternary complex. Wild-type ROQ domain was pre-incubated with labeled TNFds, and then with increasing concentrations of unlabeled Hmg19. The NaCl concentration was 50 mM. (c). EMSA results with mutations in the A site. The mutations blocked the formation of the ternary complex. (d). A model showing possible linkages between the RNA molecules in the B site and A site. A single-stranded RNA (gray, 7 nts) was modeled to connect the 3′ end of the RNA in the B site to the 5′ end of the stem-loop RNA in the A site (~30 Å distance). A linker between the 3′ end of the RNA in the A site to the 5′ end of the RNA in the B site would need to span ~60 Å (gray dots).
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Related In: Results  -  Collection

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Figure 5: Two separate RNA binding sites in the ROQ domain(a). Overlay of the structures of the Hmg19 complex (in color) and TNF23 complex (gray) of the ROQ domain, based on domains II and III. A 30° change in the orientation of domain I is indicated with the red arrow. (b). EMSA results showing the formation of the ROQ-TNFds-Hmg19 ternary complex. Wild-type ROQ domain was pre-incubated with labeled TNFds, and then with increasing concentrations of unlabeled Hmg19. The NaCl concentration was 50 mM. (c). EMSA results with mutations in the A site. The mutations blocked the formation of the ternary complex. (d). A model showing possible linkages between the RNA molecules in the B site and A site. A single-stranded RNA (gray, 7 nts) was modeled to connect the 3′ end of the RNA in the B site to the 5′ end of the stem-loop RNA in the A site (~30 Å distance). A linker between the 3′ end of the RNA in the A site to the 5′ end of the RNA in the B site would need to span ~60 Å (gray dots).
Mentions: The structures of the Hmg19 and TNF23 complexes demonstrate that the ROQ domain has two separate RNA binding sites, with no overlaps between them (Fig. 5a). The binding mode of Hmg19 RNA in the A site clearly indicates that a dsRNA cannot be accommodated there, as the loop makes intimate contacts with the ROQ domain (Fig. 2c). Our mutagenesis and EMSA results confirm that the A site in domain III is for binding stem-loop RNA, while the B site at the interface of domains I and II is for binding dsRNA.

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