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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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Functional study of Roquin in human 293T cells(a). mRNA levels as measured by quantitative real-time PCR. Roquin and Roquin-2 mRNA levels in 293T cells are shown after control knockdown (ConKD), Roquin/Roquin-2 double knockdown (Roquin1/2KD) as indicated above the figure, and complementation by add back with shRNA-resistant Roquin (WT), and Roquin (A site) and Roquin (B site) mutants. Complementation with the vector plasmid lacking the Roquin cDNA is denoted (Vector). Error bars, s.d. (n=3 cell cultures) (b). Plot showing the stability of the HMGXB3, IL-6 and PPP1R10 mRNAs. Degradation of the mRNAs was measured by quantitative real-time PCR in the same 293T cells as in panel a. Level of mRNA remaining following actinomycin D-directed transcriptional silencing at the indicated times is shown. Error bars, s.d. (n=3 cell cultures). Half-lives (t1/2) are denoted in the figure and presented relative to GAPDH mRNA and derived from three independent experiments. Comparison of the various complementation data relative to the control are indicated by the bars, and p-values from comparison of the decay rates are presented with asterisks (* p<0.05, ** p<0.01, *** p<0.001, from two-tailed extra sum-of-squares F test).
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Figure 6: Functional study of Roquin in human 293T cells(a). mRNA levels as measured by quantitative real-time PCR. Roquin and Roquin-2 mRNA levels in 293T cells are shown after control knockdown (ConKD), Roquin/Roquin-2 double knockdown (Roquin1/2KD) as indicated above the figure, and complementation by add back with shRNA-resistant Roquin (WT), and Roquin (A site) and Roquin (B site) mutants. Complementation with the vector plasmid lacking the Roquin cDNA is denoted (Vector). Error bars, s.d. (n=3 cell cultures) (b). Plot showing the stability of the HMGXB3, IL-6 and PPP1R10 mRNAs. Degradation of the mRNAs was measured by quantitative real-time PCR in the same 293T cells as in panel a. Level of mRNA remaining following actinomycin D-directed transcriptional silencing at the indicated times is shown. Error bars, s.d. (n=3 cell cultures). Half-lives (t1/2) are denoted in the figure and presented relative to GAPDH mRNA and derived from three independent experiments. Comparison of the various complementation data relative to the control are indicated by the bars, and p-values from comparison of the decay rates are presented with asterisks (* p<0.05, ** p<0.01, *** p<0.001, from two-tailed extra sum-of-squares F test).

Mentions: To assess the functional significance of the two RNA binding sites in promoting decay of mRNAs targeted by Roquin, we simultaneously knocked down human Roquin and Roquin-2 in 293T cells and complemented with exogenous wild-type and mutant Roquin (Fig. 6a), which were confirmed to be stable and expressed at comparable protein levels (MZ and MK, data not shown). We tested the stability of HMGXB3 and PPP1R10 mRNAs, which were shown to be responsive to Roquin in mouse NIH3T3 cells 5, as well as the pleiotropic cytokine IL-6 mRNA which may possess a CDE (Supplementary Fig. 3). Reduction of endogenous Roquin and Roquin-2, by 70% and 90% respectively, led to the stabilization of the human HMGXB3 and IL-6 mRNAs that was reversed upon complementation with shRNA-resistant wild-type Roquin (Fig. 6b). In contrast, despite comparable levels of expression (Fig. 6a), neither the A site nor the B site mutant Roquin complementation could restore the decay of these mRNAs, demonstrating that both sites are necessary for efficient Roquin-mediated decay of target mRNAs in cells. The effect of Roquin knockdown on IL-6 stability is smaller, consistent with the presence of other mechanisms that regulate this mRNA 21. As expected, an mRNA lacking a predicted CDE, PAQR8, was not responsive to changes in Roquin levels. Surprisingly, the PPP1R10 mRNA was also nonresponsive to a reduction in Roquin levels or complementation with Roquin in human cells, indicating some functional differences between the human and mouse cells for this mRNA.


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)

Functional study of Roquin in human 293T cells(a). mRNA levels as measured by quantitative real-time PCR. Roquin and Roquin-2 mRNA levels in 293T cells are shown after control knockdown (ConKD), Roquin/Roquin-2 double knockdown (Roquin1/2KD) as indicated above the figure, and complementation by add back with shRNA-resistant Roquin (WT), and Roquin (A site) and Roquin (B site) mutants. Complementation with the vector plasmid lacking the Roquin cDNA is denoted (Vector). Error bars, s.d. (n=3 cell cultures) (b). Plot showing the stability of the HMGXB3, IL-6 and PPP1R10 mRNAs. Degradation of the mRNAs was measured by quantitative real-time PCR in the same 293T cells as in panel a. Level of mRNA remaining following actinomycin D-directed transcriptional silencing at the indicated times is shown. Error bars, s.d. (n=3 cell cultures). Half-lives (t1/2) are denoted in the figure and presented relative to GAPDH mRNA and derived from three independent experiments. Comparison of the various complementation data relative to the control are indicated by the bars, and p-values from comparison of the decay rates are presented with asterisks (* p<0.05, ** p<0.01, *** p<0.001, from two-tailed extra sum-of-squares F test).
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Related In: Results  -  Collection

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Show All Figures
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Figure 6: Functional study of Roquin in human 293T cells(a). mRNA levels as measured by quantitative real-time PCR. Roquin and Roquin-2 mRNA levels in 293T cells are shown after control knockdown (ConKD), Roquin/Roquin-2 double knockdown (Roquin1/2KD) as indicated above the figure, and complementation by add back with shRNA-resistant Roquin (WT), and Roquin (A site) and Roquin (B site) mutants. Complementation with the vector plasmid lacking the Roquin cDNA is denoted (Vector). Error bars, s.d. (n=3 cell cultures) (b). Plot showing the stability of the HMGXB3, IL-6 and PPP1R10 mRNAs. Degradation of the mRNAs was measured by quantitative real-time PCR in the same 293T cells as in panel a. Level of mRNA remaining following actinomycin D-directed transcriptional silencing at the indicated times is shown. Error bars, s.d. (n=3 cell cultures). Half-lives (t1/2) are denoted in the figure and presented relative to GAPDH mRNA and derived from three independent experiments. Comparison of the various complementation data relative to the control are indicated by the bars, and p-values from comparison of the decay rates are presented with asterisks (* p<0.05, ** p<0.01, *** p<0.001, from two-tailed extra sum-of-squares F test).
Mentions: To assess the functional significance of the two RNA binding sites in promoting decay of mRNAs targeted by Roquin, we simultaneously knocked down human Roquin and Roquin-2 in 293T cells and complemented with exogenous wild-type and mutant Roquin (Fig. 6a), which were confirmed to be stable and expressed at comparable protein levels (MZ and MK, data not shown). We tested the stability of HMGXB3 and PPP1R10 mRNAs, which were shown to be responsive to Roquin in mouse NIH3T3 cells 5, as well as the pleiotropic cytokine IL-6 mRNA which may possess a CDE (Supplementary Fig. 3). Reduction of endogenous Roquin and Roquin-2, by 70% and 90% respectively, led to the stabilization of the human HMGXB3 and IL-6 mRNAs that was reversed upon complementation with shRNA-resistant wild-type Roquin (Fig. 6b). In contrast, despite comparable levels of expression (Fig. 6a), neither the A site nor the B site mutant Roquin complementation could restore the decay of these mRNAs, demonstrating that both sites are necessary for efficient Roquin-mediated decay of target mRNAs in cells. The effect of Roquin knockdown on IL-6 stability is smaller, consistent with the presence of other mechanisms that regulate this mRNA 21. As expected, an mRNA lacking a predicted CDE, PAQR8, was not responsive to changes in Roquin levels. Surprisingly, the PPP1R10 mRNA was also nonresponsive to a reduction in Roquin levels or complementation with Roquin in human cells, indicating some functional differences between the human and mouse cells for this mRNA.

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