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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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Interactions between the Hmg19 CDE and the ROQ domain(a). Composite omit Fo-Fc electron density map at 2.9 Å resolution for the Hmg19 RNA, contoured at 3σ. (b). Interactions of the 3-nt UGU loop and the top base pair of the stem with domain III of the ROQ domain. Hydrogen bonds and ionic interactions are shown as red dashed lines. (c). Molecular surface of the ROQ domain near the binding site for the 3-nt UGU loop, showing the close contacts between domain III of the ROQ domain and the tri-loop. (d). Interactions between the 5′ arm and U1 of Hmg19 with domains III and II of the ROQ domain.
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Figure 2: Interactions between the Hmg19 CDE and the ROQ domain(a). Composite omit Fo-Fc electron density map at 2.9 Å resolution for the Hmg19 RNA, contoured at 3σ. (b). Interactions of the 3-nt UGU loop and the top base pair of the stem with domain III of the ROQ domain. Hydrogen bonds and ionic interactions are shown as red dashed lines. (c). Molecular surface of the ROQ domain near the binding site for the 3-nt UGU loop, showing the close contacts between domain III of the ROQ domain and the tri-loop. (d). Interactions between the 5′ arm and U1 of Hmg19 with domains III and II of the ROQ domain.

Mentions: We chose the Hmgxb3 CDE for our studies as it has four G-C base pairs in the stem and is therefore expected to be more stable as a stem-loop than the TNF CDE (Fig. 1b). The stem of Hmg19 contains six Watson-Crick base pairs and assumes a slightly distorted A-form RNA structure (Fig. 2a). A U1-G17 wobble at the base of the stem was proposed in an earlier report 5, but this base pair was not formed and in fact G17 is disordered (Fig. 2a). These two nucleotides are present in TNF and Hmgxb3 but are not conserved in the other CDEs (Supplementary Fig. 3).


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)

Interactions between the Hmg19 CDE and the ROQ domain(a). Composite omit Fo-Fc electron density map at 2.9 Å resolution for the Hmg19 RNA, contoured at 3σ. (b). Interactions of the 3-nt UGU loop and the top base pair of the stem with domain III of the ROQ domain. Hydrogen bonds and ionic interactions are shown as red dashed lines. (c). Molecular surface of the ROQ domain near the binding site for the 3-nt UGU loop, showing the close contacts between domain III of the ROQ domain and the tri-loop. (d). Interactions between the 5′ arm and U1 of Hmg19 with domains III and II of the ROQ domain.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Interactions between the Hmg19 CDE and the ROQ domain(a). Composite omit Fo-Fc electron density map at 2.9 Å resolution for the Hmg19 RNA, contoured at 3σ. (b). Interactions of the 3-nt UGU loop and the top base pair of the stem with domain III of the ROQ domain. Hydrogen bonds and ionic interactions are shown as red dashed lines. (c). Molecular surface of the ROQ domain near the binding site for the 3-nt UGU loop, showing the close contacts between domain III of the ROQ domain and the tri-loop. (d). Interactions between the 5′ arm and U1 of Hmg19 with domains III and II of the ROQ domain.
Mentions: We chose the Hmgxb3 CDE for our studies as it has four G-C base pairs in the stem and is therefore expected to be more stable as a stem-loop than the TNF CDE (Fig. 1b). The stem of Hmg19 contains six Watson-Crick base pairs and assumes a slightly distorted A-form RNA structure (Fig. 2a). A U1-G17 wobble at the base of the stem was proposed in an earlier report 5, but this base pair was not formed and in fact G17 is disordered (Fig. 2a). These two nucleotides are present in TNF and Hmgxb3 but are not conserved in the other CDEs (Supplementary Fig. 3).

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