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Cited by 110RNA Research and SplicingCholesterol and Lipid MetabolismRNA Interference and Gene Delivery

The DOI matches its Crossref record; 31 of the 31 references it cites were found in a registry or an index; 10 of the 31 sources its claims cite were checked for support.

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

Open access
Adachi, Shungo, Homoto, Masae, Tanaka, Rikou, Hioki, Yusaku, Murakami, Hiroshi, Suga, Hiroaki, Matsumoto, Masaki, Nakayama, Keiichi I., Hatta, Tomohisa, Iemura, Shun-ichiro, Natsume, Tohru·2014·Nucleic Acids Research
DOI 10.1093/nar/gku652PMC PMC4150769
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Abstract

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. We also show that the C-terminal regions of ZFP36L1 and ZFP36L2 are directly phosphorylated by p90 ribosomal S6 kinase, a kinase downstream of ERK, resulting in dissociation of the CCR4-NOT-deadenylase complex and stabilization of LDLR mRNA. We further demonstrate that targeted disruption of the interaction between LDLR mRNA and ZFP36L1 and ZFP36L2 using antisense oligonucleotides results in upregulation of LDLR mRNA and protein. These results indicate that ZFP36L1 and ZFP36L2 regulate LDLR protein levels downstream of ERK. Our results also show the usefulness of our method for identifying critical regulators of specific RNAs and the potency of antisense oligonucleotide-based therapeutics.

Overview

AI snapshot

Written from the paper’s full text.

TL;DR

ZFP36L1 and ZFP36L2 bind the LDLR mRNA 3′-UTR ARE1 region and recruit the CCR4-NOT deadenylase complex to destabilize LDLR mRNA; ERK–RSK signaling phosphorylates their C-termini, dissociating CCR4-NOT and stabilizing LDLR mRNA. LNA antisense oligonucleotide Oligo-L1 disrupts this interaction, increasing LDLR mRNA/protein and LDL uptake in Hep3B cells.

Key findings

  • A proteomic screen using seven Flag-tagged bait RNAs identified ZFP36L1 and ZFP36L2 as specific binders of the LDLR mRNA 3′-UTR (ARE1–3); about 400 peptides from ∼150 proteins were identified, with ∼25% common to all baits, and ZFP36L1/L2 appeared in all four LDLR MS analyses. Endogenous ZFP36L1 co-immunoprecipitated LDLR, PLK3, and VEGFA mRNAs but not β-actin mRNA.
  • ZFP36L1 and ZFP36L2 predominantly bound the ARE1 UAUUUAUU sequence. Double siRNA knockdown of ZFP36L1 and ZFP36L2 in HeLa cells increased LDLR mRNA and protein and stabilized LDLR mRNA; PMA-mediated LDLR mRNA stabilization was lost after knockdown. LNA Oligo-L1 blocked the LDLR–ZFP36L1/L2 interaction, increased LDLR mRNA and protein in HeLa and Hep3B cells, and increased DiI-LDL uptake in Hep3B cells; control oligos L2–L6 did not.
  • ERK-dependent phosphorylation was mapped to C-terminal serine-334 of ZFP36L1 and serine-493/495 of ZFP36L2 by iTRAQ MS; PMA increased these phosphorylations and U0126 reversed them. PMA inhibited ZFP36L1–CNOT7 interaction, reversed by U0126 or RSK inhibitors BI-D1870/SL0101; recombinant RSK1 directly phosphorylated GST-ZFP36L1 at serine-334 and reduced CNOT1/CNOT7 binding.
  • ZFP36L1-SASA or ZFP36L1-ΔC mutants prevented PMA-mediated stabilization of an RFP-LDLR-3′-UTR reporter. PLK3 and VEGFA mRNAs were also stabilized by PMA, whereas cMYC mRNA was not.

Methods

  • In vitro RNA-binding protein purification: seven Flag-peptide-tagged bait RNAs, including LDLR 3′-UTR fragments, stable RNAs, and c-Myc, were incubated with 293T lysates, immunoprecipitated, and analyzed by nanoscale LC/MS/MS; interactions were validated by western blot and qPCR co-immunoprecipitation.
  • Cell-based assays in HEK293T, HeLa, and Hep3B cells: siRNA knockdown (20 nM), LNA-modified antisense oligonucleotides (80 nM), actinomycin D chase with or without PMA, qPCR normalized to β-actin or Luc2, mRNA half-lives fitted by exponential decay, western blot, and DiI-LDL uptake in Hep3B cells.
  • Phosphorylation analysis: iTRAQ-based quantitative MS of Flag-ZFP36L1/L2 from mock-, PMA-, or PMA+U0126-treated 293T cells; in vitro kinase assays with recombinant RSK1/ERK1 and GST-ZFP36L1; Student’s t-test with P < 0.002, n = 3 for qPCR groups.

Limitations

  • The paper states that the mechanisms and meaning of PMA-enhanced ZFP36L1 binding to LDLR mRNA remain unknown, and whether ZFP36 family proteins are commonly regulated by these pathways or differ is an open question.
  • The paper states that UAUUUAUU is necessary but not sufficient for ZFP36L1/L2 interaction and destabilization, and that further investigation is required to predict target mRNAs in silico; off-target effects of antisense oligonucleotides are important to consider for therapeutic applications.
  • Evident from the text: no in vivo animal or human data are presented, and delivery of Oligo-L1 to the liver is not tested; the in vitro RBP purification approach may not fully capture in vivo RNA–protein interactions, although the authors argue it has advantages.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1093/nar/gku652

Cites

31 of 31 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Verification

Claims the paper makes

10 of 31 cited sources checked · 4 supported · 1 flagged · accuracy not yet measured

  • Premisecitation

    ZFP36L1 and ZFP36L2 regulate multiple target mRNAs.

    “We used RNAi-based knockdown to assess the function of ZRP36L1 and ZFP36L2, but as with many RNA-regulating proteins, ZFP36L1 and ZFP36L2 regulate multiple target mRNAs (6).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [6] Sanduja, 2011, The roles of TTP and BRF proteins in regulated mRNA decay, Wiley Interdiscip. Rev. RNA source
  • Findingown data

    Oligo-L1 stabilizes LDLR mRNA in HeLa and 293T cells.

    “We also examined the effect of Oligo-L1 on the stability of LDLR mRNA in HeLa and 293T cells using an ActD chase experiment and we found that Oligo-L1 stabilized LDLR mRNA in these cell lines (Figure 3F, Supplementary Figure S3G and H).”
    Quote in the paperSubstantial claim
  • Findingown data

    The mobility shift of Flag-ZFP36L1 from PMA-treated 293T cells was reversed by bacterial alkaline phosphatase.

    “We also found that the mobility shift of Flag-ZFP36L1, which we immunopurified from Flag-ZFP36L1-overexpressing and PMA-treated 293T cells, could be reversed by treatment with bacterial alkaline phosphatase (Figure 4B).”
    Quote in the paperSubstantial claim
  • Findingown data

    Endogenous ZFP36L1 does not interact with β-actin mRNA.

    “ZFP36L1 did not interact with β-actin mRNA (Figure 1C).”
    Quote in the paperNot a substantial claim
  • Findingown data

    CNOT7 interacts with ZFP36L1.

    “We found that CNOT7, a critical enzymatic component of CCR4-NOT deadenylase, interacts with ZFP36L1 and interestingly, this interaction is inhibited by PMA treatment and reversed by U0126 treatment, indicating that this regulation is mediated by the ERK pathway (Figure 4E).”
    Quote in the paperSubstantial claim
  • Probably not a substantial claim. 19
  • It may state two separate claims as one. 29
  • The quote only partly states this claim. 2

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Premisecitation

    PLK3 and VEGFA mRNAs are targets of ZFP36L1.

    “PLK3 and VEGFA mRNAs have recently been identified as targets of ZFP36L1 (7,8), whereas cMYC mRNA does not bind to ZFP36L1 or ZFP36L2.”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [7] Horner, 2009, Stimulation of polo-like kinase 3 mRNA decay by tristetraprolin, Mol. Cell. Biol. source

      The Discussion (page unavailable) identifies PLK3 mRNA as a physiological target of TTP (ZFP36), apparently specific to that family member in the tested fibroblasts. The Results report that PLK3 mRNA decay was not slowed by ZFP36L1 deficiency, contrary to the attributed PLK3 claim. The retrieved passages do not address VEGFA as a ZFP36L1 target. (LLM, conf=0.90)

      Checked by a reasoning model — may be wrong.

      Experimental — accuracy not yet measured
    • cites [8] Hacker, 2010, ZFP36L1 is regulated by growth factors and cytokines in keratinocytes and influences their VEGF production, Growth Factors source

  • Probably not a substantial claim.

    Premisecitation

    The CCR4-NOT deadenylase complex interacts with the C-terminus of ZFP36.

    “The CCR4-NOT deadenylase complex has recently been shown to interact with the C-terminus of ZFP36 (16,17); therefore, we next examined whether ZFP36L1 interacts with the CCR4-NOT deadenylase complex and whether this interaction is affected by PMA.”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [16] Marchese, 2010, MAPKAP kinase 2 blocks tristetraprolin-directed mRNA decay by inhibiting CAF1 deadenylase recruitment, J. Biol. Chem. source

      The section on CAF1 recruitment (Fig. 9A–B; page unavailable) reports that CAF1a and CAF1b interact with TTP, supporting an association with a deadenylase complex. The experiments use full-length TTP and do not locate the interaction at its C-terminus; the paper describes the complex as CCR4–CAF1 rather than demonstrating an interaction with CCR4–NOT as a whole. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [17] Sandler, 2011, Not1 mediates recruitment of the deadenylase Caf1 to mRNAs targeted for degradation by tristetraprolin, Nucleic Acids Res. source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Premisecitation

    The CCR4-NOT poly(A) deadenylase complex interacts with ZFP36 and ZFP36L1 and acts as an important effector complex of ZFP36-mediated mRNA-destabilization.

    “It is known to interact with ZFP36 and ZFP36L1 and to act as an important effector complex of ZFP36-mediated mRNA-destabilization (16,17).”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [16] Marchese, 2010, MAPKAP kinase 2 blocks tristetraprolin-directed mRNA decay by inhibiting CAF1 deadenylase recruitment, J. Biol. Chem. source

      The Discussion (page unavailable) identifies CCR4–CAF1 as the main deadenylase complex involved in TTP-directed deadenylation, and the recruitment section reports that CAF1 interacts with TTP (ZFP36). Our copy does not establish an interaction with ZFP36L1, so that part of the attributed claim is unsupported. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [17] Sandler, 2011, Not1 mediates recruitment of the deadenylase Caf1 to mRNAs targeted for degradation by tristetraprolin, Nucleic Acids Res. source

      The Results sections report that Not1, a scaffold of the CCR4–NOT complex, associates with tristetraprolin (TTP/ZFP36) and is required for TTP-mediated mRNA decay. The Introduction mentions related proteins BRF1 and BRF2, but our copy does not show an interaction between CCR4–NOT and ZFP36L1; that part of the claim is unsupported. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Premisecitation

    ZFP36 family members interact with AREs containing a UAUUUAUU sequence and trigger degradation of several ARE-containing mRNAs, including PLK3 and VEGFA.

    “ZFP36L1 and ZFP36L2 belong to the family of CCCH tandem zinc finger proteins (the ZFP36 family), members of which interact with AREs containing a UAUUUAUU sequence and trigger the degradation of several ARE-containing mRNAs, including PLK3 and VEGFA (7,8,18,19).”
    Quote in the paperNot a substantial claim2 of 4 sources checked
    • cites [7] Horner, 2009, Stimulation of polo-like kinase 3 mRNA decay by tristetraprolin, Mol. Cell. Biol. source

      The Abstract says ZFP36 family members bind an ARE consensus site, UUAUUUAUU, which contains the claimed UAUUUAUU sequence. The Abstract and Discussion establish that TTP promotes PLK3 mRNA decay, but the Results find no equivalent effect for ZFP36L1 or ZFP36L2 in the fibroblasts studied. No passage located in our copy supports VEGFA as a target. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [8] Hacker, 2010, ZFP36L1 is regulated by growth factors and cytokines in keratinocytes and influences their VEGF production, Growth Factors source
    • cites [18] Hudson, 2004, Recognition of the mRNA AU-rich element by the zinc finger domain of TIS11d, Nat. Struct. Mol. Biol. source
    • cites [19] Duan, 2009, cAMP-dependent posttranscriptional regulation of steroidogenic acute regulatory (STAR) protein by the zinc finger protein ZFP36L1/TIS11b, Mol. Endocrinol. source

      The paper's background section says that ZFP36-family proteins bind AU-rich elements and promote mRNA degradation, and identifies UAUUUAUU as a binding sequence. It also reports TIS11b-mediated destabilization of VEGF mRNA. Our copy does not identify PLK3 as a target (searched: PLK3, polo-like kinase 3). Page numbers were not available in the indexed passages. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Premisecitation

    KHSRP, hnRNPD and PTBP1 are LDLR-destabilizing proteins.

    “Recently, KHSRP, hnRNPD and PTBP1 have been reported to be LDLR-destabilizing proteins (5).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [5] Li, 2009, Identification of mRNA binding proteins that regulate the stability of LDL receptor mRNA through AU-rich elements, J. Lipid. Res. source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Premisecitation

    Anti-microRNA antisense oligonucleotides efficiently blocked microRNA function, thereby alleviating diseases in rodents and nonhuman primates.

    “Recently, some reports have shown that an anti-microRNA antisense oligonucleotide efficiently blocked the function of the microRNA, thereby alleviating diseases in rodents and nonhuman primates (29,30).”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [29] Elmén, 2008, LNA-mediated microRNA silencing in non-human primates, Nature source
    • cites [30] Lanford, 2010, Therapeutic silencing of microRNA-122 in primates with chronic hepatitis C virus infection, Science source

  • It may state two separate claims as one.

    Findingown data

    PMA-mediated LDLR mRNA stabilization is observed in control siRNA-transfected cells but not in cells transfected with ZFP36L1 and ZFP36L2 siRNAs.

    “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).”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    PMA treatment inhibits the interaction between CNOT7 and ZFP36L1, and this inhibition is reversed by U0126.

    “We found that CNOT7, a critical enzymatic component of CCR4-NOT deadenylase, interacts with ZFP36L1 and interestingly, this interaction is inhibited by PMA treatment and reversed by U0126 treatment, indicating that this regulation is mediated by the ERK pathway (Figure 4E).”
    • Also stated in: § Regulation of ZFP36L1 · plain
      “We show in this report that PMA treatment causes dissociation of ZFP36L1 and CNOT7, and does not cause the dissociation of LDLR and ZFP36L1/L2 (Figure 4D).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Findingown data

    Endogenous ZFP36L1 interacts with PLK3 and VEGFA mRNAs.

    “We found that endogenous ZFP36L1 interacts with LDLR mRNA, and also with PLK3 and VEGFA mRNAs, previously identified ZFP36L1-interacting mRNAs (7,8).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [7] Horner, 2009, Stimulation of polo-like kinase 3 mRNA decay by tristetraprolin, Mol. Cell. Biol. source

      Checked before — its earlier verdict is held back until a new check runs.

    • cites [8] Hacker, 2010, ZFP36L1 is regulated by growth factors and cytokines in keratinocytes and influences their VEGF production, Growth Factors source

  • It may state two separate claims as one.

    Findingown data

    The interaction between LDLR mRNA and ZFP36L1 or ZFP36L2 is blocked by Oligo-L1 but not by Oligo-L2, -L3 or -L4.

    “We found that interaction was clearly blocked by Oligo-L1, but was not affected by Oligo-L2, -L3 or -L4 (Figure 2D).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Findingown data

    Oligo-L1 has no effect on the levels of VEGFA or PLK3 mRNAs.

    “In addition, as expected, Oligo-L1 had no effect on the levels of VEGFA or PLK3 mRNAs (Supplementary Figure S3F).”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Findingown data

    Knockdown of ZFP36L1 and ZFP36L2 together results in an increase in both LDLR mRNA and protein.

    “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).”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    The ARE1-containing region of the LDLR mRNA 3′-UTR is responsible for LDLR mRNA instability and for PMA-mediated stabilization of LDLR mRNA.

    “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.”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    Oligo-L5 and Oligo-L6 cannot inhibit the interaction between LDLR mRNA and ZFP36L1, nor increase the stability of LDLR mRNA or the levels of LDLR protein in cells.

    “Neither of these oligonucleotides could inhibit the interaction between LDLR mRNA and ZFP36L1, nor could they increase the stability of LDLR mRNA or the levels of LDLR protein in cells (Supplementary Figure S3I and J)”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    ZFP36L1 and ZFP36L2 predominantly bind to the ARE1 region of the LDLR mRNA 3′-UTR and only modestly bind to the ARE2-3 region.

    “We found that ZFP36L1 and ZFP36L2 predominantly bind to the ARE1 region, and only modestly bind to the ARE2–3 region, despite the presence of the UAUUUAUU sequence (Figure 2A and C).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: ARE1 and ARE2 of the LDLR mRNA 3′-UTR are comprised of the UAUUUAUU sequence.

  • It may state two separate claims as one.

    Findingown data

    Oligo-L1 increases the levels of LDLR mRNA and protein in HeLa cells.

    “We transfected these oligonucleotides into HeLa cells and found that Oligo-L1 increased the levels of LDLR mRNA and protein (Figure 3D and E, Supplementary Figure S3E).”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    In 293T cells, PMA treatment increased phosphorylation of C-terminal serine-334 of ZFP36L1, reversed by U0126.

    “We found that phosphorylation of the C-terminal serine-334 residue of ZFP36L1 and of the C-terminal serine-493 and -495 residues of ZFP36L2 was increased upon PMA treatment, but was reversed by U0126 treatment (Figure 4C, Supplementary Figure S5A).”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    In 293T cells, PMA treatment increased phosphorylation of C-terminal serine-493 and -495 of ZFP36L2, reversed by U0126.

    “We found that phosphorylation of the C-terminal serine-334 residue of ZFP36L1 and of the C-terminal serine-493 and -495 residues of ZFP36L2 was increased upon PMA treatment, but was reversed by U0126 treatment (Figure 4C, Supplementary Figure S5A).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Findingown data

    The study identified known interactions with other bait RNAs including 7SK-CDK9 and 7SK-LARP7.

    “We also identified several known interactions with other bait RNAs including 7SK-CDK9 and 7SK-LARP7.”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Findingown data

    KHSRP binds the 3′-UTRs of IFNA1, β-actin and cMYC mRNAs; hnRNPD and PTBP1 are common to all RNA baits.

    “However, using our approach, we identified KHSRP as a binding protein for the 3′-UTRs of IFNA1, β-actin and cMYC mRNAs and hnRNPD and PTBP1 were common to all the RNA baits.”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Findingown data

    IFNA1 mRNA is stable and ZFP36L1 does not interact strongly with its 3′-UTR.

    “Interestingly, IFNA1 mRNA, which also has a UAUUUAUU sequence in its 3′-UTR, is stable in cells and ZFP36L1 does not interact strongly with the 3′-UTR of this mRNA (Figure 1B, Supplementary Table S2).”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    PMA treatment inhibits the interactions between CNOT7 and ZFP36 and between CNOT7 and ZFP36L2.

    “We examined the interaction between CNOT7 and ZFP36 or ZFP36L2 and found that ZFP36L2, in addition to ZFP36 and ZFP36L1, also interacts with CNOT7 and that the interactions between CNOT7 and ZFP36 or ZFP36L2 are inhibited by PMA treatment (Supplementary Figure S5E).”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    IFNA1 and β-actin mRNAs are stable, whereas cMYC mRNA is unstable and not stabilized by ERK signaling.

    “In spite of the binding of these proteins, IFNA1 and β-actin mRNAs were stable, whereas cMYC mRNA was unstable and was not stabilized by ERK signaling.”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Findingown data

    Oligo-L1 inhibits the interaction between ZFP36L1 and LDLR mRNA without affecting the interaction between LDLR and hnRNPD, hnRNPI or KHSRP, or the interaction of ZFP36L1 with the 3′-UTRs of PLK3 or VEGFA mRNAs.

    “(i) Oligo-L1 could inhibit the interaction between ZFP36L1 and LRLD mRNA, without affecting the interaction between LDLR and hnRNPD, hnRNPI or KHSRP, or the interaction of ZFP36L1 with the 3′-UTRs of PLK3 or VEGFA mRNAs (Supplementary Figure S2B).”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    Control oligonucleotides Oligo-L2, -L3, -L4, -L5 and -L6 could not inhibit the interaction between LDLR mRNA and ZFP36L1, and could not increase the levels of LDLR mRNA or protein in cells.

    “(v) Four control oligonucleotides, Oligo-L2, -L3, -L4, -L5 and -L6, could not inhibit the interaction between LDLR mRNA and ZFP36L1, and also could not increase the levels of LDLR mRNA or protein in cells (Figure 3D, E and G, Supplementary Figure S3E, J and L).”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    PMA stabilized PLK3 and VEGFA mRNAs, but not cMYC mRNA.

    “We found that PLK3 and VEGFA mRNAs were also stabilized by PMA, but that this was not the case for cMYC mRNA (Supplementary Figure S7).”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Premisecitation

    LDLR mRNA is unstable but is stabilized upon PMA treatment through activation of ERK.

    “LDLR mRNA is known to be unstable, but is stabilized upon phorbol 12-myristate 13-acetate (PMA) treatment through the activation of extracellular signal-regulated kinase (ERK) (4).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [4] Kong, 2004, Berberine is a novel cholesterol-lowering drug working through a unique mechanism distinct from statins, Nat. Med. source

  • Probably not a substantial claim. It may state two separate claims as one.

    Premisecitation

    The detailed mechanisms underlying the control of LDLR mRNA stability remain unknown, and the critical regulator of this control has not been identified.

    “Although some LDLR mRNA-binding proteins have been identified, the detailed mechanisms underlying the control of LDLR mRNA stability remain unknown (4,5), and the critical regulator of this control has not been identified.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [4] Kong, 2004, Berberine is a novel cholesterol-lowering drug working through a unique mechanism distinct from statins, Nat. Med. source
    • cites [5] Li, 2009, Identification of mRNA binding proteins that regulate the stability of LDL receptor mRNA through AU-rich elements, J. Lipid. Res. source

      Checked before — its earlier verdict is held back until a new check runs.

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Study design and conductnone given

    The data shown are representative of at least three independent experiments.

    “The data are representative of at least three independent experiments.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Premisecitation

    ZFP36L1 and ZFP36L2 belong to the family of CCCH tandem zinc finger proteins (the ZFP36 family).

    “ZFP36L1 and ZFP36L2 belong to the family of CCCH tandem zinc finger proteins (the ZFP36 family, which includes ZFP36, ZFP36L1 and ZFP36L2) (6).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [6] Sanduja, 2011, The roles of TTP and BRF proteins in regulated mRNA decay, Wiley Interdiscip. Rev. RNA source

      Checked before — its earlier verdict is held back until a new check runs.

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Premisecitation

    ZFP36 family proteins bind to AREs and trigger degradation of several ARE-containing mRNAs, including PLK3 and VEGFA.

    “ZFP36 family proteins bind to AREs and trigger the degradation of several ARE-containing mRNAs, including PLK3 and vascular endothelial growth factor A (VEGFA) (7,8).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [7] Horner, 2009, Stimulation of polo-like kinase 3 mRNA decay by tristetraprolin, Mol. Cell. Biol. source

      Checked before — its earlier verdict is held back until a new check runs.

    • cites [8] Hacker, 2010, ZFP36L1 is regulated by growth factors and cytokines in keratinocytes and influences their VEGF production, Growth Factors source

  • It may state two separate claims as one.

    Interpretationown data

    ZFP36L1 and ZFP36L2 are LDLR mRNA-destabilizing factors that are indispensable for PMA-mediated stabilization of LDLR mRNA.

    “These results suggest that ZFP36L1 and ZFP36L2 are LDLR mRNA-destabilizing factors that are indispensable for PMA-mediated stabilization of LDLR mRNA.”
    Quote in the paperSubstantial claim

  • The quote only partly states this claim.

    Interpretationown data

    ZFP36L1 and ZFP36L2-mediated LDLR mRNA destabilization is conserved among 293T, HeLa and Hep3B cells.

    “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).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Study design and conductown data

    The method identified well-known 7SK RBPs, including CDK9 and LARP7, as 7SK-Flag specific binding proteins, demonstrating the accuracy of the strategy.

    “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.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [14] Diribarne, 2009, 7SK RNA, a non-coding RNA regulating P-TEFb, a general transcription factor, RNA Biol. source

      Checked before — its earlier verdict is held back until a new check runs.

  • The quote only partly states this claim.

    Interpretationown data

    Phosphorylation of ZFP36L1 and ZFP36L2 by RSK causes dissociation of the CCR4-NOT-deadenylase complex and stabilization of LDLR mRNA.

    “resulting in dissociation of the CCR4-NOT-deadenylase complex and stabilization of LDLR mRNA”
    Quote in the paperSubstantial claim
    • Rests on an assumption: RSK is downstream of ERK.

  • Probably not a substantial claim. It may state two separate claims as one.

    Study design and conductcitation

    The fully automated robotic IP and protein identification system is highly sensitive and reproducible.

    “Given these advantages, plus our fully automated robotic IP and protein identification system, which is highly sensitive and reproducible (11), we opted to use the in vitro purification approach, combined with comparison analysis.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [11] Iemura, 2012, One-by-one sample preparation method for protein network analysis source

      Checked before — its earlier verdict is held back until a new check runs.

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Interpretationcitation

    Antisense oligonucleotide-based therapeutics are potent.

    “These results demonstrate the potency of antisense oligonucleotide-based therapeutics.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Premisecitation

    MAPKAPK2, a downstream kinase of p38, phosphorylates ZFP36, but not in the C-terminal region, leading to dissociation of ZFP36 and CNOT7.

    “Recently, MAPKAPK2, a downstream kinase of p38, has been shown to phosphorylate ZFP36, but not in the C-terminal region, leading to dissociation of ZFP36 and CNOT7 (16,17).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [16] Marchese, 2010, MAPKAP kinase 2 blocks tristetraprolin-directed mRNA decay by inhibiting CAF1 deadenylase recruitment, J. Biol. Chem. source

      Checked before — its earlier verdict is held back until a new check runs.

    • cites [17] Sandler, 2011, Not1 mediates recruitment of the deadenylase Caf1 to mRNAs targeted for degradation by tristetraprolin, Nucleic Acids Res. source

      Checked before — its earlier verdict is held back until a new check runs.

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Premisecitation

    Antisense oligonucleotides allow elucidation of the binding sites and the function of RNA/RBP interactions in cells.

    “As Cibois et al. have also reported (27), antisense oligonucleotides allow us to elucidate the binding sites and the function of RNA/RBP interactions in cells.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [27] Cibois, 2010, A strategy to analyze the phenotypic consequences of inhibiting the association of an RNA-binding protein with a specific RNA, RNA source

      Checked before — its earlier verdict is held back until a new check runs.

  • It may state two separate claims as one.

    Interpretationown data

    The UAUUUAUU sequence is necessary but not sufficient for ZFP36L1 and ZFP36L2 to interact with and destabilize mRNA.

    “Our results suggest that the UAUUUAUU sequence is necessary but not sufficient for ZFP36L1 and ZFP36L2 to interact with and destabilize mRNA.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim.

    Premisecitation

    Increasing the amount of LDLR protein in the liver could be a therapeutic approach for hyperlipidemia.

    “It has been reported that increasing the amount of LDLR protein in the liver could be a therapeutic approach for hyperlipidemia (28).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [28] Garg, 2007, Update on dyslipidemia, J. Clin. Endocrinol. Metab. source
41 of 41

  • The paper relies on this without support.

    RSK is downstream of ERK.

    “p90 ribosomal S6 kinase, a kinase downstream of ERK”

  • The paper relies on this without support.

    ARE1 and ARE2 of the LDLR mRNA 3′-UTR are comprised of the UAUUUAUU sequence.

    “ARE1 and ARE2 are comprised of the UAUUUAUU sequence.”

  • The paper relies on this without support.

    The UAUUUAUU sequence is the consensus binding sequence for ZFP36L1 and ZFP36L2.

    “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 paper relies on this without support.

    LDLR in the liver binds LDL and thereby lowers blood LDL levels.

    “In the liver, the LDLR binds to LDL, thereby lowering blood LDL levels.”

  • The paper relies on this without support.

    cMYC mRNA does not bind to ZFP36L1 or ZFP36L2.

    “whereas cMYC mRNA does not bind to ZFP36L1 or ZFP36L2.”
5 of 5

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • CSDE1, hnRNPD, hnRNPI, KSRP, ZFP36L1, and ZFP36L2 bind to AREs in the 3′UTR and lead to degradation of LDLR mRNA, decreasing LDLR.

    “CSDE1, hnRNPD, hnRNPI, KSRP, ZFP36L1, ZFP36L2 Bind to AREs in the 3′UTR and lead to degradation of LDLR mRNA ↓ [29-31]”

    From The low-density lipoprotein receptor: Emerging post-transcriptional regulatory mechanisms (2025) · cites this paper as [30]

    The Results and Discussion (pages not provided) show that ZFP36L1 and ZFP36L2 bind the LDLR 3′-UTR ARE1 region, destabilize LDLR mRNA, and reduce LDLR protein levels. The Discussion reports KHSRP, hnRNPD and PTBP1 as LDLR-destabilizing proteins, but does not establish that each binds an LDLR ARE. Our copy does not identify CSDE1 as an LDLR-binding or destabilizing protein. (LLM, conf=0.88)

    Experimental — accuracy not yet measured

  • Only the ERK-dependent phosphorylation of endogenous ZFP36L1 has been rigorously examined.

    “only the ERK-dependent phosphorylation of endogenous ZFP36L1 has been rigorously examined [15]”

    From Triciribine Engages ZFP36L1 and HuR to Stabilize LDLR mRNA (2020) · cites this paper as [15]

    In Results, ‘ZFP36L1 is regulated by phosphorylation downstream of ERK’ (page unavailable), the authors report phosphorylation of endogenous ZFP36L1 at serine-334 after PMA treatment. They establish ERK-pathway dependence using tagged ZFP36L1, but the cited passage does not establish ERK dependence specifically for endogenous protein or the broader claim that only this phosphorylation has been rigorously examined. (LLM, conf=0.84)

    Experimental — accuracy not yet measured

  • HuR and ZFP36L1 were considered likely candidates because they have been reported to modulate LDLR mRNA stability in response to agents that activate ERK and their mechanisms have been partially resolved.

    “These two proteins were considered to be likely candidates, because (1) they have been reported to modulate LDLR mRNA stability in response to agents that activate ERK and (2) the mechanisms underlying their regulatory effect on LDLR mRNA stability have been partially resolved [9,13,14,15].”

    From Triciribine Engages ZFP36L1 and HuR to Stabilize LDLR mRNA (2020) · cites this paper as [15]

    The Introduction and Discussion report that ZFP36L1 destabilizes LDLR mRNA and that ERK–RSK signaling inhibits this activity; the Introduction also describes phosphorylation of ZFP36L1 as part of the mechanism. These passages support the claim for ZFP36L1, but our copy does not establish the corresponding claim for HuR. Page numbers were not provided. (LLM, conf=0.85)

    Experimental — accuracy not yet measured

  • ZFP36L1 and ZFP36L2 influence LDLR mRNA stability, operate in an ERK-dependent manner, bind to the 3′UTR of LDLR mRNA, and promote its destabilization.

    “Using a proteomics-based approach, two additional RBPs, ZFP36L1 and ZFP36L2, that influence LDLR mRNA stability were identified [30].These proteins operate in an ERK-dependent manner, binding to the 3′UTR of LDLR mRNA and promoting its destabilization.”

    From The low-density lipoprotein receptor: Emerging post-transcriptional regulatory mechanisms (2025) · cites this paper as [30]

  • PMA increased LDLR mRNA stability through phosphorylation of ZFP36L1 and its subsequent release from CNOT7.

    “Adachi et al. demonstrated that PMA increased LDLR mRNA stability through the phosphorylation of ZFP36L1 and its subsequent release from CNOT7 [15].”

    From Triciribine Engages ZFP36L1 and HuR to Stabilize LDLR mRNA (2020) · cites this paper as [15]

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Five control oligonucleotides are called four

    Minor

    The sentence says there are four control oligonucleotides but names five: L2, L3, L4, L5 and L6.

  • Figure 4B names different phosphatases

    Minor

    The results text says the experiment used bacterial alkaline phosphatase, while the Figure 4B caption says it used bacterial acid phosphatase.

  • Figure 4H cell line differs between results and caption

    Minor

    The results describe the Figure 4H ActD chase experiment as being performed in 293T cells, but its caption identifies HeLa cells.

3 of 3

Citations

Cites

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Metadata

Authors
Adachi, S., Homoto, M., Tanaka, R., Hioki, Y., Murakami, H., Suga, H., Matsumoto, M., Nakayama, K. I., Hatta, T., Iemura, S.-I., & Natsume, T.
Affiliations
  • National Institute of Advanced Industrial Science and Technology (JP)
  • The University of Tokyo (JP)
  • Kyushu University (JP)
Corresponding author
Natsume, Tohru
ORCID
Venue
Nucleic Acids Research
Publisher
Oxford University Press (OUP)
Published
2014-08-08
Volume
42
Issue
15
Pages
10037-10049
ISSN
  • 0305-1048 (ISSN-L)
  • 1362-4962
DOI
10.1093/nar/gku652
PMC
PMC4150769
OpenAlex
W2019768782
Cited by
109
References
30
Citations by year
2026: 9 · 2025: 10 · 2024: 20 · 2023: 9 · 2022: 7 · 2021: 16 · 2020: 9 · 2019: 8 · 2018: 6 · 2017: 3 · 2016: 3 · 2015: 8 · 2014: 1
FWCI
1.65
Type
journal article
Language
en
Open access
Licence
Locations
  • Nucleic Acids Research (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Funders
  • National Institute of Advanced Industrial Science and Technology
  • Japan Society for the Promotion of Science (26640080, 24115001)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Research and Splicing
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biochemistry › Cholesterol and Lipid Metabolism
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
Keywords
ZFP36L1, ZFP36L2, LDL receptor, mRNA stability, ERK signaling, p90 ribosomal S6 kinase, CCR4-NOT complex, mRNA deadenylation, 3' untranslated region, protein phosphorylation, antisense oligonucleotides, lipid metabolism
MeSH terms
Cell Line, HeLa Cells, Humans, Phosphorylation, Receptors, LDL, RNA, Messenger, Transcription Factors, 3' Untranslated Regions, RNA Stability, MAP Kinase Signaling System, Ribosomal Protein S6 Kinases, 90-kDa, Extracellular Signal-Regulated MAP Kinases, Butyrate Response Factor 1, HEK293 Cells

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

A language model drew the claims from the paper on 2026-11-09, under version 3 of the claim definition (model deepseek-v4.1-flash, prompt extract-v3).

A second model labelled each claim on 2026-11-09 (model jev-1.13-20260917, definition revision gold-guideline-v0.2).

Cited sources are checked with a language model’s help (gpt-6-sol).

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

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