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