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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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Identification of bait-specific RBPs. (A) Flow chart summarizing the experiment. (B) Confirmation of our results by western blot analysis. 293T cells were lysed in lysis buffer and the cleared lysates were subjected to IP with anti-Flag antibody in combination with the indicated bait RNAs. Co-immunoprecipitated RNA and proteins were eluted using the Flag-peptide and were then subjected to western blot analysis using the indicated antibodies. Five percent of the initial amount of cleared 293T lysate was loaded as total lysate. An IP experiment without bait RNA was performed as a control. (C) Confirmation of the endogenous interaction between ZFP36L1 and LDLR mRNA. 293T cells were lysed in lysis buffer and the cleared lysates were subjected to IP with control or anti-ZFP36L1 antibody. Total RNA and co-immunoprecipitated RNA were extracted, and quantitative reverse-transcription (RT)-PCR (qPCR) was performed using primers specific to LDLR, PLK3, VEGFA and β-actin mRNAs. Results are shown as% of input. The data are representative of at least three independent experiments. Error bars show standard deviation of the mean.
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Figure 1: Identification of bait-specific RBPs. (A) Flow chart summarizing the experiment. (B) Confirmation of our results by western blot analysis. 293T cells were lysed in lysis buffer and the cleared lysates were subjected to IP with anti-Flag antibody in combination with the indicated bait RNAs. Co-immunoprecipitated RNA and proteins were eluted using the Flag-peptide and were then subjected to western blot analysis using the indicated antibodies. Five percent of the initial amount of cleared 293T lysate was loaded as total lysate. An IP experiment without bait RNA was performed as a control. (C) Confirmation of the endogenous interaction between ZFP36L1 and LDLR mRNA. 293T cells were lysed in lysis buffer and the cleared lysates were subjected to IP with control or anti-ZFP36L1 antibody. Total RNA and co-immunoprecipitated RNA were extracted, and quantitative reverse-transcription (RT)-PCR (qPCR) was performed using primers specific to LDLR, PLK3, VEGFA and β-actin mRNAs. Results are shown as% of input. The data are representative of at least three independent experiments. Error bars show standard deviation of the mean.

Mentions: To identify the critical protein controlling the stability of LDLR mRNA, we first developed the method of Flag-peptide-tagging the 3′-end of in vitro transcribed RNA (Supplementary Figure S1A and B; see Experimental Procedures). We then validated whether Flag-peptide-tagged RNA can be used to co-immunoprecipitate its binding protein, using HA-tagged-MS2 and a Flag-peptide-tagged-RNA that contains an MS2-binding site (13) (Supplementary Figure S1C). We found that Flag-peptide-tagged RNA can be used for co-immunoprecipitation of its binding protein (Supplementary Figure S1D). Next, we hypothesized that the critical protein controlling LDLR mRNA stability would bind specifically to its 3′-UTR region, but would not bind to stable mRNAs or unstable mRNAs that are not stabilized by PMA treatment. We then selected seven bait RNAs, including LDLR mRNA, five stable RNAs (β-actin mRNA, IFNA1 mRNA, MBP mRNA, hnRNP A2/B1 mRNAs and 7SK RNA) and one very unstable mRNA, c-Myc, which is not stabilized by PMA treatment (Table S1). We synthesized these RNAs in vitro and conjugated a Flag-peptide to their 3′-ends. We performed an IP experiment using these seven bait RNAs and a 293T cell lysate. The co-immunoprecipitated proteins were eluted using the Flag peptide, and then digested with lysyl endopeptidase, and all peptides obtained were directly analyzed by MS (Figure 1A). For each RNA, we conducted two independent IP experiments and performed MS analysis in duplicate to obtain four sets of data. We identified about 400 kinds of peptides derived from ∼150 proteins (Table S2). Approximately 25% of these proteins, including IGF2BP1, were common to all the RNA baits. We then extracted the LDLR mRNA-specific binding proteins that were only identified in all four MS analyses of LDLR samples and found ZFP36L1 and ZFP36L2 as proteins that bind specifically to the LDLR mRNA 3′-UTR (ARE1–3) (Table S3). Using this method, we also found well-known 7SK RBPs, including CDK9 and LARP7 as 7SK-Flag specific binding porteins (14) (Table S4). This result demonstrates the accuracy of our strategy. We confirmed the interactions of bait RNAs and their specific binding proteins by western blotting (Figure 1B). To further confirm the endogenous interaction between LDLR mRNA and ZFP36L1, we performed a co-immunoprecipitation experiment using the antibody against ZFP36L1 and 293T cell lysate. We found that endogenous ZFP36L1 interacts with LDLR mRNA, and also with PLK3 and VEGFA mRNAs, previously identified ZFP36L1-interacting mRNAs (7,8). ZFP36L1 did not interact with β-actin mRNA (Figure 1C).


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)

Identification of bait-specific RBPs. (A) Flow chart summarizing the experiment. (B) Confirmation of our results by western blot analysis. 293T cells were lysed in lysis buffer and the cleared lysates were subjected to IP with anti-Flag antibody in combination with the indicated bait RNAs. Co-immunoprecipitated RNA and proteins were eluted using the Flag-peptide and were then subjected to western blot analysis using the indicated antibodies. Five percent of the initial amount of cleared 293T lysate was loaded as total lysate. An IP experiment without bait RNA was performed as a control. (C) Confirmation of the endogenous interaction between ZFP36L1 and LDLR mRNA. 293T cells were lysed in lysis buffer and the cleared lysates were subjected to IP with control or anti-ZFP36L1 antibody. Total RNA and co-immunoprecipitated RNA were extracted, and quantitative reverse-transcription (RT)-PCR (qPCR) was performed using primers specific to LDLR, PLK3, VEGFA and β-actin mRNAs. Results are shown as% of input. The data are representative of at least three independent experiments. Error bars show standard deviation of the mean.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4150769&req=5

Figure 1: Identification of bait-specific RBPs. (A) Flow chart summarizing the experiment. (B) Confirmation of our results by western blot analysis. 293T cells were lysed in lysis buffer and the cleared lysates were subjected to IP with anti-Flag antibody in combination with the indicated bait RNAs. Co-immunoprecipitated RNA and proteins were eluted using the Flag-peptide and were then subjected to western blot analysis using the indicated antibodies. Five percent of the initial amount of cleared 293T lysate was loaded as total lysate. An IP experiment without bait RNA was performed as a control. (C) Confirmation of the endogenous interaction between ZFP36L1 and LDLR mRNA. 293T cells were lysed in lysis buffer and the cleared lysates were subjected to IP with control or anti-ZFP36L1 antibody. Total RNA and co-immunoprecipitated RNA were extracted, and quantitative reverse-transcription (RT)-PCR (qPCR) was performed using primers specific to LDLR, PLK3, VEGFA and β-actin mRNAs. Results are shown as% of input. The data are representative of at least three independent experiments. Error bars show standard deviation of the mean.
Mentions: To identify the critical protein controlling the stability of LDLR mRNA, we first developed the method of Flag-peptide-tagging the 3′-end of in vitro transcribed RNA (Supplementary Figure S1A and B; see Experimental Procedures). We then validated whether Flag-peptide-tagged RNA can be used to co-immunoprecipitate its binding protein, using HA-tagged-MS2 and a Flag-peptide-tagged-RNA that contains an MS2-binding site (13) (Supplementary Figure S1C). We found that Flag-peptide-tagged RNA can be used for co-immunoprecipitation of its binding protein (Supplementary Figure S1D). Next, we hypothesized that the critical protein controlling LDLR mRNA stability would bind specifically to its 3′-UTR region, but would not bind to stable mRNAs or unstable mRNAs that are not stabilized by PMA treatment. We then selected seven bait RNAs, including LDLR mRNA, five stable RNAs (β-actin mRNA, IFNA1 mRNA, MBP mRNA, hnRNP A2/B1 mRNAs and 7SK RNA) and one very unstable mRNA, c-Myc, which is not stabilized by PMA treatment (Table S1). We synthesized these RNAs in vitro and conjugated a Flag-peptide to their 3′-ends. We performed an IP experiment using these seven bait RNAs and a 293T cell lysate. The co-immunoprecipitated proteins were eluted using the Flag peptide, and then digested with lysyl endopeptidase, and all peptides obtained were directly analyzed by MS (Figure 1A). For each RNA, we conducted two independent IP experiments and performed MS analysis in duplicate to obtain four sets of data. We identified about 400 kinds of peptides derived from ∼150 proteins (Table S2). Approximately 25% of these proteins, including IGF2BP1, were common to all the RNA baits. We then extracted the LDLR mRNA-specific binding proteins that were only identified in all four MS analyses of LDLR samples and found ZFP36L1 and ZFP36L2 as proteins that bind specifically to the LDLR mRNA 3′-UTR (ARE1–3) (Table S3). Using this method, we also found well-known 7SK RBPs, including CDK9 and LARP7 as 7SK-Flag specific binding porteins (14) (Table S4). This result demonstrates the accuracy of our strategy. We confirmed the interactions of bait RNAs and their specific binding proteins by western blotting (Figure 1B). To further confirm the endogenous interaction between LDLR mRNA and ZFP36L1, we performed a co-immunoprecipitation experiment using the antibody against ZFP36L1 and 293T cell lysate. We found that endogenous ZFP36L1 interacts with LDLR mRNA, and also with PLK3 and VEGFA mRNAs, previously identified ZFP36L1-interacting mRNAs (7,8). ZFP36L1 did not interact with β-actin mRNA (Figure 1C).

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