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

In the paper

“PLK3 and VEGFA mRNAs have recently been identified as targets of ZFP36L1 (7,8), whereas cMYC mRNA does not bind to ZFP36L1 or ZFP36L2.”

What the sources say

• Disputed

  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.

  cites [7] Horner, 2009, Stimulation of polo-like kinase 3 mRNA decay by tristetraprolin, Mol. Cell. Biol. sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites [8] Hacker, 2010, ZFP36L1 is regulated by growth factors and cytokines in keratinocytes and influences their VEGF production, Growth Factors source

In the paper

“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.”

What the sources say

• Partly supported

  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)

  cites [16] Marchese, 2010, MAPKAP kinase 2 blocks tristetraprolin-directed mRNA decay by inhibiting CAF1 deadenylase recruitment, J. Biol. Chem. sourceExperimental — accuracy not yet measured
• Supported

  The Abstract states that Not1, a scaffold of the Ccr4–Not complex, associates with the C-terminal domain of tristetraprolin (TTP/ZFP36). The Results section, “The C-terminal domain of TTP is critical for recruitment of Not1 and Caf1” (page not provided), also reports interaction with Not1, Caf1a and Ccr4a through that domain. (LLM, conf=0.93)

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

In the paper

“We found that endogenous ZFP36L1 interacts with LDLR mRNA, and also with PLK3 and VEGFA mRNAs, previously identified ZFP36L1-interacting mRNAs (7,8).”

What the sources say

• ?Could not verify

  The Results and Discussion report that Plk3 mRNA decay was unchanged in ZFP36L1-deficient fibroblasts and identify Plk3 as a physiological target of TTP in that setting. Those findings do not test whether endogenous ZFP36L1 physically interacts with Plk3 mRNA. No passage found addresses an interaction between ZFP36L1 and VEGFA mRNA (searched: ZFP36L1 binding, VEGFA, VEGF). (LLM, conf=0.75)

  cites [7] Horner, 2009, Stimulation of polo-like kinase 3 mRNA decay by tristetraprolin, Mol. Cell. Biol. sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites [8] Hacker, 2010, ZFP36L1 is regulated by growth factors and cytokines in keratinocytes and influences their VEGF production, Growth Factors source

In the paper

“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).”

What the sources say

• Partly supported

  The Discussion (page not provided) establishes that TTP/ZFP36 binds AREs in PLK3 mRNA and promotes its decay. It also reports that PLK3 mRNA was not stabilized in fibroblasts lacking the related proteins ZFP36L1 or ZFP36L2. No passage in our copy supports VEGFA as a target, so the full list attributed to this paper is not supported. (LLM, conf=0.88)

  cites [7] Horner, 2009, Stimulation of polo-like kinase 3 mRNA decay by tristetraprolin, Mol. Cell. Biol. sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites [8] Hacker, 2010, ZFP36L1 is regulated by growth factors and cytokines in keratinocytes and influences their VEGF production, Growth Factors source

In the paper

“It is known to interact with ZFP36 and ZFP36L1 and to act as an important effector complex of ZFP36-mediated mRNA-destabilization (16,17).”

What the sources say

• Partly supported

  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)

  cites [16] Marchese, 2010, MAPKAP kinase 2 blocks tristetraprolin-directed mRNA decay by inhibiting CAF1 deadenylase recruitment, J. Biol. Chem. sourceExperimental — accuracy not yet measured
• Partly supported

  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)

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

In the paper

“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).”

What the sources say

• Partly supported

  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)

  cites [7] Horner, 2009, Stimulation of polo-like kinase 3 mRNA decay by tristetraprolin, Mol. Cell. Biol. sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites [8] Hacker, 2010, ZFP36L1 is regulated by growth factors and cytokines in keratinocytes and influences their VEGF production, Growth Factors source
• ?Not checked — no full text of the sourcecites [18] Hudson, 2004, Recognition of the mRNA AU-rich element by the zinc finger domain of TIS11d, Nat. Struct. Mol. Biol. source
• Partly supported

  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)

  cites [19] Duan, 2009, cAMP-dependent posttranscriptional regulation of steroidogenic acute regulatory (STAR) protein by the zinc finger protein ZFP36L1/TIS11b, Mol. Endocrinol. sourceExperimental — accuracy not yet measured

In the paper

“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.”

What the sources say

• Partly supported

  The concluding section reports that the automated one-by-one system enabled reproducible identification of protein interaction partners. The Discussion reports improved signal-to-noise ratio and more known interactors, but the paper presents highly sensitive analyses as an expectation, not an established performance result. (LLM, conf=0.85)

  cites [11] Iemura, 2012, One-by-one sample preparation method for protein network analysis sourceExperimental — accuracy not yet measured

In the paper

“Recently, KHSRP, hnRNPD and PTBP1 have been reported to be LDLR-destabilizing proteins (5).”

What the sources say

• Supported

  The Abstract states that hnRNP D, hnRNP I and KSRP act as negative regulators of LDLR mRNA stability. The Results also report increased LDLR mRNA after depletion of all three. These are the proteins named in the claim as hnRNPD, PTBP1 and KHSRP, respectively. (LLM, conf=0.93)

  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

In the paper

“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).”

What the sources say

• Partly supported

  The paper identifies MK2 as a downstream kinase of p38 and reports that its phosphorylation of TTP (ZFP36) reduces binding to CAF1a (CNOT7) by about 50% (introduction and ‘Recruitment of CAF1 to TTP’ section; page unavailable). It identifies Ser-52 and Ser-178 as necessary for the effect, but our copy does not establish that phosphorylation excludes the C-terminal region. The reported reduction in binding also does not establish complete dissociation. (LLM, conf=0.86)

  cites [16] Marchese, 2010, MAPKAP kinase 2 blocks tristetraprolin-directed mRNA decay by inhibiting CAF1 deadenylase recruitment, J. Biol. Chem. sourceExperimental — accuracy not yet measured
• Partly supported

  The Discussion (page unavailable) says MK2 phosphorylates TTP/ZFP36 at S52 and S178 and that phosphorylation reduces its interaction with Caf1; the Introduction identifies Caf1a as CNOT7. Our copy does not establish that phosphorylation excludes the C-terminal region or identify MK2 as downstream of p38. It reports reduced interaction, not complete dissociation. (LLM, conf=0.86)

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

In the paper

“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).”

What the sources say

• ?Not checked — no full text of the sourcecites [29] Elmén, 2008, LNA-mediated microRNA silencing in non-human primates, Nature source
• Supported

  The paper’s treatment results report that an oligonucleotide complementary to miR-122 antagonized miR-122 in chimpanzees with chronic HCV infection. The Abstract reports sustained suppression of viremia and improvement of HCV-induced liver pathology. This supports the nonhuman-primate part of the jointly cited sentence; the rodent part is not attributed to this paper alone. (LLM, conf=0.91)

  cites [30] Lanford, 2010, Therapeutic silencing of microRNA-122 in primates with chronic hepatitis C virus infection, Science source

In the paper

“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).”

In the paper

“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).”

In the paper

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

In the paper

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

In the paper

“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).”

In the paper

“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.”

In the paper

“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)”

In the paper

“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).”

In the paper

“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).”

In the paper

“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).”

In the paper

“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).”

In the paper

“We also identified several known interactions with other bait RNAs including 7SK-CDK9 and 7SK-LARP7.”

In the paper

“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.”

In the paper

“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).”

In the paper

“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).”

In the paper

“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.”

In the paper

“(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).”

In the paper

“(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).”

In the paper

“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).”

In the paper

“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).”

What the sources say

• ?Not checked — no full text of the sourcecites [4] Kong, 2004, Berberine is a novel cholesterol-lowering drug working through a unique mechanism distinct from statins, Nat. Med. source

In the paper

“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.”

What the sources say

• ?Not checked — no full text of the sourcecites [4] Kong, 2004, Berberine is a novel cholesterol-lowering drug working through a unique mechanism distinct from statins, Nat. Med. source
• Not checked yet

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

  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

In the paper

“The data are representative of at least three independent experiments.”

In the paper

“ZFP36L1 and ZFP36L2 belong to the family of CCCH tandem zinc finger proteins (the ZFP36 family, which includes ZFP36, ZFP36L1 and ZFP36L2) (6).”

What the sources say

• Not checked yet

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

  cites [6] Sanduja, 2011, The roles of TTP and BRF proteins in regulated mRNA decay, Wiley Interdiscip. Rev. RNA source

In the paper

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

In the paper

“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).”

In the paper

“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.”

What the sources say

• Not checked yet

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

  cites [14] Diribarne, 2009, 7SK RNA, a non-coding RNA regulating P-TEFb, a general transcription factor, RNA Biol. source

In the paper

“resulting in dissociation of the CCR4-NOT-deadenylase complex and stabilization of LDLR mRNA”

In the paper

“These results demonstrate the potency of antisense oligonucleotide-based therapeutics.”

In the paper

“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.”

What the sources say

• Not checked yet

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

  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

In the paper

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

In the paper

“It has been reported that increasing the amount of LDLR protein in the liver could be a therapeutic approach for hyperlipidemia (28).”

What the sources say

• ?Not checked — no full text of the sourcecites [28] Garg, 2007, Update on dyslipidemia, J. Clin. Endocrinol. Metab. source