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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.

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Domain analysis of the 3′-UTR of LDLR mRNA. (A) Schematic representation of the 3′-UTR of LDLR mRNA. Black bars indicate the regions used for ActD chase and IP experiments. AREs 1–3 are indicated by red boxes and the UCAU repeat is indicated by the white box. Designed oligonucleotides are indicated by blue lines. Conservation around the LDLR ARE1 region is shown in the blue graph, created using Vertebrate Multiz Alignment & PhastCons Conservation (28 species), as extracted from the UCSC Human Genome Browser (http://genome.ucsc.edu/). The consensus ZFP36L1- and ZFP36L2-binding sequence is colored in red. Sequences of the designed oligonucleotides, L1 and L2, are indicated. (B) RFP-tagged LDLR 3′-UTR constructs were expressed in 293T cells, and cells were then subjected to ActD chase experiments. Twenty-four hours after transfection, 293T cells were treated with ActD and PMA as indicated (PMA treatment commenced 10 min after ActD treatment). qPCR was performed using RFP and Luc2-specific primers. Results were normalized to the levels of Luc2 mRNA. The data are representative of at least three independent experiments. mRNA half-lives were calculated by fitting an exponential decay curve to all time points. Figures in the graph indicate the mean value of the mRNA decay half-lives. Error bars show standard deviation of the mean. P-values were calculated using Student's t-test. *P < 0.002; n = 3 for each group. (C) 293T cells were transfected with Myc-tagged ZFP36L1 and ZFP36L2. Cells were then lysed with lysis buffer and the cleared lysates were subjected to IP with the indicated LDLR 3′-UTR bait RNAs. Immunoprecipitated proteins were subjected to western blot analysis with the anti-Myc antibody. (D) 293T cells were transfected with Myc-tagged ZFP36L1 and ZFP36L2. Cells were then lysed with lysis buffer and the cleared lysates were subjected to IP with the indicated LDLR 3′-UTR bait RNAs and the indicated oligos. Immunoprecipitated proteins were subjected to western blot analysis with the anti-Myc antibody. These data are representative of at least three independent experiments.
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Figure 2: Domain analysis of the 3′-UTR of LDLR mRNA. (A) Schematic representation of the 3′-UTR of LDLR mRNA. Black bars indicate the regions used for ActD chase and IP experiments. AREs 1–3 are indicated by red boxes and the UCAU repeat is indicated by the white box. Designed oligonucleotides are indicated by blue lines. Conservation around the LDLR ARE1 region is shown in the blue graph, created using Vertebrate Multiz Alignment & PhastCons Conservation (28 species), as extracted from the UCSC Human Genome Browser (http://genome.ucsc.edu/). The consensus ZFP36L1- and ZFP36L2-binding sequence is colored in red. Sequences of the designed oligonucleotides, L1 and L2, are indicated. (B) RFP-tagged LDLR 3′-UTR constructs were expressed in 293T cells, and cells were then subjected to ActD chase experiments. Twenty-four hours after transfection, 293T cells were treated with ActD and PMA as indicated (PMA treatment commenced 10 min after ActD treatment). qPCR was performed using RFP and Luc2-specific primers. Results were normalized to the levels of Luc2 mRNA. The data are representative of at least three independent experiments. mRNA half-lives were calculated by fitting an exponential decay curve to all time points. Figures in the graph indicate the mean value of the mRNA decay half-lives. Error bars show standard deviation of the mean. P-values were calculated using Student's t-test. *P < 0.002; n = 3 for each group. (C) 293T cells were transfected with Myc-tagged ZFP36L1 and ZFP36L2. Cells were then lysed with lysis buffer and the cleared lysates were subjected to IP with the indicated LDLR 3′-UTR bait RNAs. Immunoprecipitated proteins were subjected to western blot analysis with the anti-Myc antibody. (D) 293T cells were transfected with Myc-tagged ZFP36L1 and ZFP36L2. Cells were then lysed with lysis buffer and the cleared lysates were subjected to IP with the indicated LDLR 3′-UTR bait RNAs and the indicated oligos. Immunoprecipitated proteins were subjected to western blot analysis with the anti-Myc antibody. These data are representative of at least three independent experiments.

Mentions: ZFP36L1 and ZFP36L2 are known as proteins that bind to a certain type of ARE that contains the sequence UAUUUAUU, causing destabilization of target mRNAs. The LDLR mRNA 3′-UTR contains three AREs (ARE1–3) (4); ARE1 and ARE2 are comprised of the UAUUUAUU sequence. We investigated the region responsible for LDLR mRNA instability and PMA-mediated stabilization (PMA is an activator of ERK) in an ActD chase experiment. After transfection of 293T cells with the reporter constructs, RFP-LDLR-3′-UTR-ARE1–3, RFP-LDLR-3′-UTR-ARE2–3 or RFP-LDLR-3′-UTR-ARE1, we examined the stability of the reporter mRNA and the effect of PMA on stability using quantitative reverse-transcription (RT)-PCR (qPCR) analysis. We also calculated the half-life of each RFP-reporter mRNA (Figure 2A and B). We found that the ARE1-containing region is not only responsible for LDLR mRNA instability, but is also responsible for PMA-mediated stabilization of LDLR mRNA.


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)

Domain analysis of the 3′-UTR of LDLR mRNA. (A) Schematic representation of the 3′-UTR of LDLR mRNA. Black bars indicate the regions used for ActD chase and IP experiments. AREs 1–3 are indicated by red boxes and the UCAU repeat is indicated by the white box. Designed oligonucleotides are indicated by blue lines. Conservation around the LDLR ARE1 region is shown in the blue graph, created using Vertebrate Multiz Alignment & PhastCons Conservation (28 species), as extracted from the UCSC Human Genome Browser (http://genome.ucsc.edu/). The consensus ZFP36L1- and ZFP36L2-binding sequence is colored in red. Sequences of the designed oligonucleotides, L1 and L2, are indicated. (B) RFP-tagged LDLR 3′-UTR constructs were expressed in 293T cells, and cells were then subjected to ActD chase experiments. Twenty-four hours after transfection, 293T cells were treated with ActD and PMA as indicated (PMA treatment commenced 10 min after ActD treatment). qPCR was performed using RFP and Luc2-specific primers. Results were normalized to the levels of Luc2 mRNA. The data are representative of at least three independent experiments. mRNA half-lives were calculated by fitting an exponential decay curve to all time points. Figures in the graph indicate the mean value of the mRNA decay half-lives. Error bars show standard deviation of the mean. P-values were calculated using Student's t-test. *P < 0.002; n = 3 for each group. (C) 293T cells were transfected with Myc-tagged ZFP36L1 and ZFP36L2. Cells were then lysed with lysis buffer and the cleared lysates were subjected to IP with the indicated LDLR 3′-UTR bait RNAs. Immunoprecipitated proteins were subjected to western blot analysis with the anti-Myc antibody. (D) 293T cells were transfected with Myc-tagged ZFP36L1 and ZFP36L2. Cells were then lysed with lysis buffer and the cleared lysates were subjected to IP with the indicated LDLR 3′-UTR bait RNAs and the indicated oligos. Immunoprecipitated proteins were subjected to western blot analysis with the anti-Myc antibody. These data are representative of at least three independent experiments.
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Figure 2: Domain analysis of the 3′-UTR of LDLR mRNA. (A) Schematic representation of the 3′-UTR of LDLR mRNA. Black bars indicate the regions used for ActD chase and IP experiments. AREs 1–3 are indicated by red boxes and the UCAU repeat is indicated by the white box. Designed oligonucleotides are indicated by blue lines. Conservation around the LDLR ARE1 region is shown in the blue graph, created using Vertebrate Multiz Alignment & PhastCons Conservation (28 species), as extracted from the UCSC Human Genome Browser (http://genome.ucsc.edu/). The consensus ZFP36L1- and ZFP36L2-binding sequence is colored in red. Sequences of the designed oligonucleotides, L1 and L2, are indicated. (B) RFP-tagged LDLR 3′-UTR constructs were expressed in 293T cells, and cells were then subjected to ActD chase experiments. Twenty-four hours after transfection, 293T cells were treated with ActD and PMA as indicated (PMA treatment commenced 10 min after ActD treatment). qPCR was performed using RFP and Luc2-specific primers. Results were normalized to the levels of Luc2 mRNA. The data are representative of at least three independent experiments. mRNA half-lives were calculated by fitting an exponential decay curve to all time points. Figures in the graph indicate the mean value of the mRNA decay half-lives. Error bars show standard deviation of the mean. P-values were calculated using Student's t-test. *P < 0.002; n = 3 for each group. (C) 293T cells were transfected with Myc-tagged ZFP36L1 and ZFP36L2. Cells were then lysed with lysis buffer and the cleared lysates were subjected to IP with the indicated LDLR 3′-UTR bait RNAs. Immunoprecipitated proteins were subjected to western blot analysis with the anti-Myc antibody. (D) 293T cells were transfected with Myc-tagged ZFP36L1 and ZFP36L2. Cells were then lysed with lysis buffer and the cleared lysates were subjected to IP with the indicated LDLR 3′-UTR bait RNAs and the indicated oligos. Immunoprecipitated proteins were subjected to western blot analysis with the anti-Myc antibody. These data are representative of at least three independent experiments.
Mentions: ZFP36L1 and ZFP36L2 are known as proteins that bind to a certain type of ARE that contains the sequence UAUUUAUU, causing destabilization of target mRNAs. The LDLR mRNA 3′-UTR contains three AREs (ARE1–3) (4); ARE1 and ARE2 are comprised of the UAUUUAUU sequence. We investigated the region responsible for LDLR mRNA instability and PMA-mediated stabilization (PMA is an activator of ERK) in an ActD chase experiment. After transfection of 293T cells with the reporter constructs, RFP-LDLR-3′-UTR-ARE1–3, RFP-LDLR-3′-UTR-ARE2–3 or RFP-LDLR-3′-UTR-ARE1, we examined the stability of the reporter mRNA and the effect of PMA on stability using quantitative reverse-transcription (RT)-PCR (qPCR) analysis. We also calculated the half-life of each RFP-reporter mRNA (Figure 2A and B). We found that the ARE1-containing region is not only responsible for LDLR mRNA instability, but is also responsible for PMA-mediated stabilization of LDLR mRNA.

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