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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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Biochemical characterizations of the ROQ domain-RNA interactions(a). Electrophoretic mobility shift assay (EMSA) results for wild-type and mutant ROQ domains with Hmg19, TNF23 and TNFds RNAs. The NaCl concentration was 200 mM. (b). EMSA results at 50 mM NaCl concentration. Please see Supplementary Fig. 7 for the uncropped images of the middle panel.
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Figure 4: Biochemical characterizations of the ROQ domain-RNA interactions(a). Electrophoretic mobility shift assay (EMSA) results for wild-type and mutant ROQ domains with Hmg19, TNF23 and TNFds RNAs. The NaCl concentration was 200 mM. (b). EMSA results at 50 mM NaCl concentration. Please see Supplementary Fig. 7 for the uncropped images of the middle panel.

Mentions: The Hmg19 and TNF23 RNAs were incubated with the wild-type and mutant ROQ domain proteins in the presence of 200 mM NaCl. The EMSA results showed that mutations in the A site blocked binding to both Hmg19 and TNF23, while mutations in the B site had only small effects on binding of the two RNAs (Fig. 4a). These results confirm the structural observations and suggest that both Hmg19 and TNF23 predominantly adopt the stem-loop conformation under these experimental conditions (1 μM RNA concentration).


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)

Biochemical characterizations of the ROQ domain-RNA interactions(a). Electrophoretic mobility shift assay (EMSA) results for wild-type and mutant ROQ domains with Hmg19, TNF23 and TNFds RNAs. The NaCl concentration was 200 mM. (b). EMSA results at 50 mM NaCl concentration. Please see Supplementary Fig. 7 for the uncropped images of the middle panel.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Biochemical characterizations of the ROQ domain-RNA interactions(a). Electrophoretic mobility shift assay (EMSA) results for wild-type and mutant ROQ domains with Hmg19, TNF23 and TNFds RNAs. The NaCl concentration was 200 mM. (b). EMSA results at 50 mM NaCl concentration. Please see Supplementary Fig. 7 for the uncropped images of the middle panel.
Mentions: The Hmg19 and TNF23 RNAs were incubated with the wild-type and mutant ROQ domain proteins in the presence of 200 mM NaCl. The EMSA results showed that mutations in the A site blocked binding to both Hmg19 and TNF23, while mutations in the B site had only small effects on binding of the two RNAs (Fig. 4a). These results confirm the structural observations and suggest that both Hmg19 and TNF23 predominantly adopt the stem-loop conformation under these experimental conditions (1 μM RNA concentration).

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