Limits...
ZFP36L1 and ZFP36L2 control LDLR mRNA stability via the ERK-RSK pathway.

Adachi S, Homoto M, Tanaka R, Hioki Y, Murakami H, Suga H, Matsumoto M, Nakayama KI, Hatta T, Iemura S, Natsume T - Nucleic Acids Res. (2014)

Bottom Line: Low-density lipoprotein receptor (LDLR) mRNA is unstable, but is stabilized upon extracellular signal-regulated kinase (ERK) activation, possibly through the binding of certain proteins to the LDLR mRNA 3'-untranslated region (UTR), although the detailed mechanism underlying this stability control is unclear.Here, using a proteomic approach, we show that proteins ZFP36L1 and ZFP36L2 specifically bind to the 3'-UTR of LDLR mRNA and recruit the CCR4-NOT-deadenylase complex, resulting in mRNA destabilization.These results indicate that ZFP36L1 and ZFP36L2 regulate LDLR protein levels downstream of ERK.

View Article: PubMed Central - PubMed

Affiliation: Molecular Profiling Research Center for Drug Discovery (molprof), National Institute of Advanced Industrial Science and Technology (AIST), Tokyo 135-0064, Japan Galaxy Pharma Inc., Akita 010-0951, Japan.

Show MeSH

Related in: MedlinePlus

LDLR mRNA is destabilized by ZFP36L1 and ZFP36L2. (A) HeLa cells were transfected with ZFP36L1 and ZFP36L2 siRNAs. Forty-eight hours after transfection, cells were harvested, total RNA was extracted and quantitative RT-PCR (qPCR) was performed using primers specific to LDLR mRNA and β-actin mRNA. Results were normalized to β-actin mRNA levels. Error bars show standard deviation of the mean. P-values against control were calculated using Student's t-test.*P < 0.002; n = 3 for each group. (B) Forty-eight hours after transfection, cells were harvested and the lysates were subjected to western blot analysis using the indicated antibodies. (C) Forty-eight hours after transfection, cells were treated with ActD and chased for the indicated time with or without PMA (PMA treatment commenced 10 min after ActD treatment). Total RNA was extracted and qPCR was performed using primers specific to LDLR mRNA and β-actin mRNA. Results were normalized to the levels of β-actin mRNA. Error bars show standard deviation of the mean. (D) HeLa cells were transfected with the indicated oligos. Twenty-four hours after transfection, cells were harvested and total RNA was extracted. Quantitative RT-PCR (qPCR) was performed using primers specific for LDLR and β-actin. Results were normalized to β-actin mRNA levels. Error bars show standard deviation of the mean. P-values against control were calculated using Student's t-test. *P < 0.002; n = 3 for each group. (E) HeLa cells were transfected with the indicated oligos. Twenty-four hours after transfection, cells were harvested and the lysates were subjected to western blot analysis using the indicated antibodies. (F) HeLa cells were transfected with the indicated oligos. Twenty-four hours after transfection, cells were treated with ActD and chased for the indicated times. Total RNA was extracted and qPCR was performed using primers specific to LDLR mRNA and β-actin mRNA. Results were normalized to the levels of β-actin mRNA. Error bars show standard deviation of the mean. (G) Hep3B cells were transfected with the indicated oligos. Twenty-four hours after transfection, cells were harvested and the lysates were subjected to western blot analysis using the indicated antibodies. (H) Hep3B cells were transfected with the indicated oligos. Twenty-four hours after transfection, cells were treated with DiI-LDL for 1 h. The cells were then lysed in RIPA buffer and the ratio of DiI-LDL fluorescence/protein concentration was measured. Error bars show standard deviation of the mean. P-values against control were calculated using Student's t-test. *P < 0.002; n = 3 for each group. The data are representative of at least three independent experiments. The number below the figure indicates the number of times we replicated the experiment. Data from one of the independent experiments are shown in Supplementary Figure S4A–H.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4150769&req=5

Figure 3: LDLR mRNA is destabilized by ZFP36L1 and ZFP36L2. (A) HeLa cells were transfected with ZFP36L1 and ZFP36L2 siRNAs. Forty-eight hours after transfection, cells were harvested, total RNA was extracted and quantitative RT-PCR (qPCR) was performed using primers specific to LDLR mRNA and β-actin mRNA. Results were normalized to β-actin mRNA levels. Error bars show standard deviation of the mean. P-values against control were calculated using Student's t-test.*P < 0.002; n = 3 for each group. (B) Forty-eight hours after transfection, cells were harvested and the lysates were subjected to western blot analysis using the indicated antibodies. (C) Forty-eight hours after transfection, cells were treated with ActD and chased for the indicated time with or without PMA (PMA treatment commenced 10 min after ActD treatment). Total RNA was extracted and qPCR was performed using primers specific to LDLR mRNA and β-actin mRNA. Results were normalized to the levels of β-actin mRNA. Error bars show standard deviation of the mean. (D) HeLa cells were transfected with the indicated oligos. Twenty-four hours after transfection, cells were harvested and total RNA was extracted. Quantitative RT-PCR (qPCR) was performed using primers specific for LDLR and β-actin. Results were normalized to β-actin mRNA levels. Error bars show standard deviation of the mean. P-values against control were calculated using Student's t-test. *P < 0.002; n = 3 for each group. (E) HeLa cells were transfected with the indicated oligos. Twenty-four hours after transfection, cells were harvested and the lysates were subjected to western blot analysis using the indicated antibodies. (F) HeLa cells were transfected with the indicated oligos. Twenty-four hours after transfection, cells were treated with ActD and chased for the indicated times. Total RNA was extracted and qPCR was performed using primers specific to LDLR mRNA and β-actin mRNA. Results were normalized to the levels of β-actin mRNA. Error bars show standard deviation of the mean. (G) Hep3B cells were transfected with the indicated oligos. Twenty-four hours after transfection, cells were harvested and the lysates were subjected to western blot analysis using the indicated antibodies. (H) Hep3B cells were transfected with the indicated oligos. Twenty-four hours after transfection, cells were treated with DiI-LDL for 1 h. The cells were then lysed in RIPA buffer and the ratio of DiI-LDL fluorescence/protein concentration was measured. Error bars show standard deviation of the mean. P-values against control were calculated using Student's t-test. *P < 0.002; n = 3 for each group. The data are representative of at least three independent experiments. The number below the figure indicates the number of times we replicated the experiment. Data from one of the independent experiments are shown in Supplementary Figure S4A–H.

Mentions: To investigate the significance of the interaction between LDLR mRNA and ZFP36L1 or ZFP36L2, we performed double siRNA-mediated knockdown of ZFP36L1 and ZFP36L2. We used HeLa cells because siRNA-mediated gene silencing is more efficient in HeLa cells than in HEK293T cells (15). We first examined the efficiency of knockdown using qPCR (Supplementary Figure S3A). We then examined the effect of this knockdown on LDLR mRNA and protein levels. We found that knockdown of ZFP36L1 and ZFP36L2 together resulted in an increase in both LDLR mRNA and protein (Figure 3A and B, Supplementary Figure S3B). Next, we examined the effect of siRNA on LDLR mRNA stability using an ActD chase experiment. We found that LDLR mRNA in cells transfected with ZFP36L1 and ZFP36L2 siRNA was clearly more stabilized than that in control siRNA-transfected cells (Figure 3C, Supplementary Figure S3C). We also observed PMA-mediated LDLR mRNA stabilization in cells transfected with control siRNA but, interestingly, not in cells transfected with ZFP36L1 and ZFP36L2 siRNAs (Figure 3C, Supplementary Figure S3C). These results suggest that ZFP36L1 and ZFP36L2 are LDLR mRNA-destabilizing factors that are indispensable for PMA-mediated stabilization of LDLR mRNA. ZFP36L1 and ZFP36L2-mediated LDLR mRNA destabilization was also observed in other cell lines, including 293T cells and Hep3B cells, indicating that this regulation is conserved among these cells (Supplementary Figure S3D).


ZFP36L1 and ZFP36L2 control LDLR mRNA stability via the ERK-RSK pathway.

Adachi S, Homoto M, Tanaka R, Hioki Y, Murakami H, Suga H, Matsumoto M, Nakayama KI, Hatta T, Iemura S, Natsume T - Nucleic Acids Res. (2014)

LDLR mRNA is destabilized by ZFP36L1 and ZFP36L2. (A) HeLa cells were transfected with ZFP36L1 and ZFP36L2 siRNAs. Forty-eight hours after transfection, cells were harvested, total RNA was extracted and quantitative RT-PCR (qPCR) was performed using primers specific to LDLR mRNA and β-actin mRNA. Results were normalized to β-actin mRNA levels. Error bars show standard deviation of the mean. P-values against control were calculated using Student's t-test.*P < 0.002; n = 3 for each group. (B) Forty-eight hours after transfection, cells were harvested and the lysates were subjected to western blot analysis using the indicated antibodies. (C) Forty-eight hours after transfection, cells were treated with ActD and chased for the indicated time with or without PMA (PMA treatment commenced 10 min after ActD treatment). Total RNA was extracted and qPCR was performed using primers specific to LDLR mRNA and β-actin mRNA. Results were normalized to the levels of β-actin mRNA. Error bars show standard deviation of the mean. (D) HeLa cells were transfected with the indicated oligos. Twenty-four hours after transfection, cells were harvested and total RNA was extracted. Quantitative RT-PCR (qPCR) was performed using primers specific for LDLR and β-actin. Results were normalized to β-actin mRNA levels. Error bars show standard deviation of the mean. P-values against control were calculated using Student's t-test. *P < 0.002; n = 3 for each group. (E) HeLa cells were transfected with the indicated oligos. Twenty-four hours after transfection, cells were harvested and the lysates were subjected to western blot analysis using the indicated antibodies. (F) HeLa cells were transfected with the indicated oligos. Twenty-four hours after transfection, cells were treated with ActD and chased for the indicated times. Total RNA was extracted and qPCR was performed using primers specific to LDLR mRNA and β-actin mRNA. Results were normalized to the levels of β-actin mRNA. Error bars show standard deviation of the mean. (G) Hep3B cells were transfected with the indicated oligos. Twenty-four hours after transfection, cells were harvested and the lysates were subjected to western blot analysis using the indicated antibodies. (H) Hep3B cells were transfected with the indicated oligos. Twenty-four hours after transfection, cells were treated with DiI-LDL for 1 h. The cells were then lysed in RIPA buffer and the ratio of DiI-LDL fluorescence/protein concentration was measured. Error bars show standard deviation of the mean. P-values against control were calculated using Student's t-test. *P < 0.002; n = 3 for each group. The data are representative of at least three independent experiments. The number below the figure indicates the number of times we replicated the experiment. Data from one of the independent experiments are shown in Supplementary Figure S4A–H.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4150769&req=5

Figure 3: LDLR mRNA is destabilized by ZFP36L1 and ZFP36L2. (A) HeLa cells were transfected with ZFP36L1 and ZFP36L2 siRNAs. Forty-eight hours after transfection, cells were harvested, total RNA was extracted and quantitative RT-PCR (qPCR) was performed using primers specific to LDLR mRNA and β-actin mRNA. Results were normalized to β-actin mRNA levels. Error bars show standard deviation of the mean. P-values against control were calculated using Student's t-test.*P < 0.002; n = 3 for each group. (B) Forty-eight hours after transfection, cells were harvested and the lysates were subjected to western blot analysis using the indicated antibodies. (C) Forty-eight hours after transfection, cells were treated with ActD and chased for the indicated time with or without PMA (PMA treatment commenced 10 min after ActD treatment). Total RNA was extracted and qPCR was performed using primers specific to LDLR mRNA and β-actin mRNA. Results were normalized to the levels of β-actin mRNA. Error bars show standard deviation of the mean. (D) HeLa cells were transfected with the indicated oligos. Twenty-four hours after transfection, cells were harvested and total RNA was extracted. Quantitative RT-PCR (qPCR) was performed using primers specific for LDLR and β-actin. Results were normalized to β-actin mRNA levels. Error bars show standard deviation of the mean. P-values against control were calculated using Student's t-test. *P < 0.002; n = 3 for each group. (E) HeLa cells were transfected with the indicated oligos. Twenty-four hours after transfection, cells were harvested and the lysates were subjected to western blot analysis using the indicated antibodies. (F) HeLa cells were transfected with the indicated oligos. Twenty-four hours after transfection, cells were treated with ActD and chased for the indicated times. Total RNA was extracted and qPCR was performed using primers specific to LDLR mRNA and β-actin mRNA. Results were normalized to the levels of β-actin mRNA. Error bars show standard deviation of the mean. (G) Hep3B cells were transfected with the indicated oligos. Twenty-four hours after transfection, cells were harvested and the lysates were subjected to western blot analysis using the indicated antibodies. (H) Hep3B cells were transfected with the indicated oligos. Twenty-four hours after transfection, cells were treated with DiI-LDL for 1 h. The cells were then lysed in RIPA buffer and the ratio of DiI-LDL fluorescence/protein concentration was measured. Error bars show standard deviation of the mean. P-values against control were calculated using Student's t-test. *P < 0.002; n = 3 for each group. The data are representative of at least three independent experiments. The number below the figure indicates the number of times we replicated the experiment. Data from one of the independent experiments are shown in Supplementary Figure S4A–H.
Mentions: To investigate the significance of the interaction between LDLR mRNA and ZFP36L1 or ZFP36L2, we performed double siRNA-mediated knockdown of ZFP36L1 and ZFP36L2. We used HeLa cells because siRNA-mediated gene silencing is more efficient in HeLa cells than in HEK293T cells (15). We first examined the efficiency of knockdown using qPCR (Supplementary Figure S3A). We then examined the effect of this knockdown on LDLR mRNA and protein levels. We found that knockdown of ZFP36L1 and ZFP36L2 together resulted in an increase in both LDLR mRNA and protein (Figure 3A and B, Supplementary Figure S3B). Next, we examined the effect of siRNA on LDLR mRNA stability using an ActD chase experiment. We found that LDLR mRNA in cells transfected with ZFP36L1 and ZFP36L2 siRNA was clearly more stabilized than that in control siRNA-transfected cells (Figure 3C, Supplementary Figure S3C). We also observed PMA-mediated LDLR mRNA stabilization in cells transfected with control siRNA but, interestingly, not in cells transfected with ZFP36L1 and ZFP36L2 siRNAs (Figure 3C, Supplementary Figure S3C). These results suggest that ZFP36L1 and ZFP36L2 are LDLR mRNA-destabilizing factors that are indispensable for PMA-mediated stabilization of LDLR mRNA. ZFP36L1 and ZFP36L2-mediated LDLR mRNA destabilization was also observed in other cell lines, including 293T cells and Hep3B cells, indicating that this regulation is conserved among these cells (Supplementary Figure S3D).

Bottom Line: Low-density lipoprotein receptor (LDLR) mRNA is unstable, but is stabilized upon extracellular signal-regulated kinase (ERK) activation, possibly through the binding of certain proteins to the LDLR mRNA 3'-untranslated region (UTR), although the detailed mechanism underlying this stability control is unclear.Here, using a proteomic approach, we show that proteins ZFP36L1 and ZFP36L2 specifically bind to the 3'-UTR of LDLR mRNA and recruit the CCR4-NOT-deadenylase complex, resulting in mRNA destabilization.These results indicate that ZFP36L1 and ZFP36L2 regulate LDLR protein levels downstream of ERK.

View Article: PubMed Central - PubMed

Affiliation: Molecular Profiling Research Center for Drug Discovery (molprof), National Institute of Advanced Industrial Science and Technology (AIST), Tokyo 135-0064, Japan Galaxy Pharma Inc., Akita 010-0951, Japan.

Show MeSH
Related in: MedlinePlus