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Staufen1-mediated mRNA decay induces Requiem mRNA decay through binding of Staufen1 to the Requiem 3'UTR.

Kim MY, Park J, Lee JJ, Ha DH, Kim J, Kim CG, Hwang J, Kim CG - Nucleic Acids Res. (2014)

Bottom Line: By screening a complementary deoxyribonucleic acid (cDNA) expression library with an RNA-ligand binding assay, we identified STAU1 as an interactor of the REQ mRNA 3'UTR.Specifically, we provide evidence that STAU1 binds to putative 30-nucleotide stem-loop-structured RNA sequences within the G8 region, which we term the protein binding site core; this binding triggers the degradation of REQ mRNA and thus regulates translation.Furthermore, we demonstrate that siRNA-mediated silencing of either STAU1 or UPF1 increases the abundance of cellular REQ mRNA and, consequently, the REQ protein, indicating that REQ mRNA is a target of SMD.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Science and Research Institute for Natural Sciences, College of Natural Sciences.

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STAU1-mediated mRNA decay (SMD) regulates cellular REQ mRNA decay. (A) HeLa cells were transiently transfected with the indicated siRNAs, and cell lysates were then subjected to western blot analysis using the specified antibodies. PLCγ served as an internal control to account for variations in protein loading. The three leftmost lanes show 3-fold dilutions of control siRNA-transfected HeLa cell lysates, demonstrating that the western blot conditions are semi-quantitative. Mean value and standard deviation (SD) were calculated from four independent experiments. (B) Total cellular RNA was purified from the cells used in (A), and RT-qPCR was performed. The mRNA levels of REQ, c-Jun and IL-7R (the latter two were used as positive controls for SMD) were normalized to the level of GAPDH mRNA. (C) HeLa cells were transiently transfected with control, UPF1 or STAU1 siRNA. After 3 days, DRB was added (100 μg/ml), and cells were incubated for the indicated time periods. Total RNA was then isolated and analyzed by RT-qPCR. Columns (B) or points (C) and error bars represent the mean and standard deviation from at least three independent experiments. The numbers above the bars indicate the mean values from the experiments.
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Figure 5: STAU1-mediated mRNA decay (SMD) regulates cellular REQ mRNA decay. (A) HeLa cells were transiently transfected with the indicated siRNAs, and cell lysates were then subjected to western blot analysis using the specified antibodies. PLCγ served as an internal control to account for variations in protein loading. The three leftmost lanes show 3-fold dilutions of control siRNA-transfected HeLa cell lysates, demonstrating that the western blot conditions are semi-quantitative. Mean value and standard deviation (SD) were calculated from four independent experiments. (B) Total cellular RNA was purified from the cells used in (A), and RT-qPCR was performed. The mRNA levels of REQ, c-Jun and IL-7R (the latter two were used as positive controls for SMD) were normalized to the level of GAPDH mRNA. (C) HeLa cells were transiently transfected with control, UPF1 or STAU1 siRNA. After 3 days, DRB was added (100 μg/ml), and cells were incubated for the indicated time periods. Total RNA was then isolated and analyzed by RT-qPCR. Columns (B) or points (C) and error bars represent the mean and standard deviation from at least three independent experiments. The numbers above the bars indicate the mean values from the experiments.

Mentions: If STAU1 binds to the REQ 3′UTR, it is plausible that endogenous REQ mRNA is degraded by SMD. To test this hypothesis, we employed HeLa cells, which exhibit higher transfection efficiency than either MEL or K562 cells and have been used in many studies of SMD. We transiently transfected HeLa cells with siRNA targeting either STAU1 and UPF1 (both essential factors in mammalian SMD) or a nonspecific control siRNA (Figure 5A and B). Western blot analysis revealed that siRNA-mediated silencing of UPF1 and STAU1 reduced the levels of these proteins to ∼20% and 10%, respectively, of the amounts found in control cells (the levels of UPF1 and STAU1 were also normalized to the level of PLCγ in order to control variations in protein loading) (Figure 5A). Moreover, silencing of UPF1 did not affect the level of STAU1, and silencing of STAU1 did not affect the level of UPF1 (Figure 5A). When UPF1 and STAU1 were individually depleted, the abundance of endogenous REQ was upregulated by ∼1.5- and 1.7-fold, respectively. These results suggest that SMD reduces REQ expression by degradation of REQ mRNA.


Staufen1-mediated mRNA decay induces Requiem mRNA decay through binding of Staufen1 to the Requiem 3'UTR.

Kim MY, Park J, Lee JJ, Ha DH, Kim J, Kim CG, Hwang J, Kim CG - Nucleic Acids Res. (2014)

STAU1-mediated mRNA decay (SMD) regulates cellular REQ mRNA decay. (A) HeLa cells were transiently transfected with the indicated siRNAs, and cell lysates were then subjected to western blot analysis using the specified antibodies. PLCγ served as an internal control to account for variations in protein loading. The three leftmost lanes show 3-fold dilutions of control siRNA-transfected HeLa cell lysates, demonstrating that the western blot conditions are semi-quantitative. Mean value and standard deviation (SD) were calculated from four independent experiments. (B) Total cellular RNA was purified from the cells used in (A), and RT-qPCR was performed. The mRNA levels of REQ, c-Jun and IL-7R (the latter two were used as positive controls for SMD) were normalized to the level of GAPDH mRNA. (C) HeLa cells were transiently transfected with control, UPF1 or STAU1 siRNA. After 3 days, DRB was added (100 μg/ml), and cells were incubated for the indicated time periods. Total RNA was then isolated and analyzed by RT-qPCR. Columns (B) or points (C) and error bars represent the mean and standard deviation from at least three independent experiments. The numbers above the bars indicate the mean values from the experiments.
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Related In: Results  -  Collection

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Figure 5: STAU1-mediated mRNA decay (SMD) regulates cellular REQ mRNA decay. (A) HeLa cells were transiently transfected with the indicated siRNAs, and cell lysates were then subjected to western blot analysis using the specified antibodies. PLCγ served as an internal control to account for variations in protein loading. The three leftmost lanes show 3-fold dilutions of control siRNA-transfected HeLa cell lysates, demonstrating that the western blot conditions are semi-quantitative. Mean value and standard deviation (SD) were calculated from four independent experiments. (B) Total cellular RNA was purified from the cells used in (A), and RT-qPCR was performed. The mRNA levels of REQ, c-Jun and IL-7R (the latter two were used as positive controls for SMD) were normalized to the level of GAPDH mRNA. (C) HeLa cells were transiently transfected with control, UPF1 or STAU1 siRNA. After 3 days, DRB was added (100 μg/ml), and cells were incubated for the indicated time periods. Total RNA was then isolated and analyzed by RT-qPCR. Columns (B) or points (C) and error bars represent the mean and standard deviation from at least three independent experiments. The numbers above the bars indicate the mean values from the experiments.
Mentions: If STAU1 binds to the REQ 3′UTR, it is plausible that endogenous REQ mRNA is degraded by SMD. To test this hypothesis, we employed HeLa cells, which exhibit higher transfection efficiency than either MEL or K562 cells and have been used in many studies of SMD. We transiently transfected HeLa cells with siRNA targeting either STAU1 and UPF1 (both essential factors in mammalian SMD) or a nonspecific control siRNA (Figure 5A and B). Western blot analysis revealed that siRNA-mediated silencing of UPF1 and STAU1 reduced the levels of these proteins to ∼20% and 10%, respectively, of the amounts found in control cells (the levels of UPF1 and STAU1 were also normalized to the level of PLCγ in order to control variations in protein loading) (Figure 5A). Moreover, silencing of UPF1 did not affect the level of STAU1, and silencing of STAU1 did not affect the level of UPF1 (Figure 5A). When UPF1 and STAU1 were individually depleted, the abundance of endogenous REQ was upregulated by ∼1.5- and 1.7-fold, respectively. These results suggest that SMD reduces REQ expression by degradation of REQ mRNA.

Bottom Line: By screening a complementary deoxyribonucleic acid (cDNA) expression library with an RNA-ligand binding assay, we identified STAU1 as an interactor of the REQ mRNA 3'UTR.Specifically, we provide evidence that STAU1 binds to putative 30-nucleotide stem-loop-structured RNA sequences within the G8 region, which we term the protein binding site core; this binding triggers the degradation of REQ mRNA and thus regulates translation.Furthermore, we demonstrate that siRNA-mediated silencing of either STAU1 or UPF1 increases the abundance of cellular REQ mRNA and, consequently, the REQ protein, indicating that REQ mRNA is a target of SMD.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Science and Research Institute for Natural Sciences, College of Natural Sciences.

Show MeSH
Related in: MedlinePlus