BackgroundTurnover of mRNA is a critical step in the regulation of gene expression, and an important step in mRNA decay is removal of the 5′ cap. We previously demonstrated that the expression of some immediate early gene mRNAs is controlled by RNA stability during early differentiation of 3T3-L1 preadipocytes.Methodology/Principal FindingsHere we show that the mouse decapping protein Dcp1a is phosphorylated via the ERK signaling pathway during early differentiation of preadipocytes. Mass spectrometry analysis and site-directed mutagenesis combined with a kinase assay identified ERK pathway–mediated dual phosphorylation at Ser 315 and Ser 319 of Dcp1a. To understand the functional effects of Dcp1a phosphorylation, we examined protein-protein interactions between Dcp1a and other decapping components with co-immunoprecipitation. Dcp1a interacted with Ddx6 and Edc3 through its proline-rich C-terminal extension, whereas the conserved EVH1 (enabled vasodilator-stimulated protein homology 1) domain in the N terminus of Dcp1a showed a stronger interaction with Dcp2. Once ERK signaling was activated, the interaction between Dcp1a and Ddx6, Edc3, or Edc4 was not affected by Dcp1a phosphorylation. Phosphorylated Dcp1a did, however, enhanced interaction with Dcp2. Protein complexes immunoprecipitated with the recombinant phosphomimetic Dcp1a(S315D/S319D) mutant contained more Dcp2 than did those immunoprecipitated with the nonphosphorylated Dcp1a(S315A/S319A) mutant. In addition, Dcp1a associated with AU-rich element (ARE)-containing mRNAs such as MAPK phosphatase-1 (MKP-1), whose mRNA stability was analyzed under the overexpression of Dcp1a constructs in the Dcp1a knockdown 3T3-L1 cells.Conclusions/SignificanceOur findings suggest that ERK-phosphorylated Dcp1a enhances its interaction with the decapping enzyme Dcp2 during early differentiation of 3T3-L1 cells.

BackgroundThe tristetraprolin (TTP) family of mRNA-binding proteins contains three major members, Ttp, Zfp36l1, and Zfp36l2. Ttp down-regulates the stability of AU-rich element–containing mRNAs and functions as an anti-inflammation regulator.MethodsTo examine whether other TTP family proteins also play roles in the inflammatory response, their expression profiles and the possible mRNA targets were determined in the knockdown cells.ResultsTtp mRNA and protein were highly induced by lipopolysaccharide (LPS), whereas Zfp36l1 and Zfp36l2 mRNAs were down-regulated and their proteins were phosphorylated during early lipopolysaccharide stimulation. Biochemical and functional analyses exhibited that the decrease of Zfp36l2 mRNA was cross-regulated by Ttp. Knockdown of Zfp36l1 and Zfp36l2 increased the basal level of Mkp-1 mRNAs by prolonging its half-life. Increasing the expression of Mkp-1 inhibited the activation of p38 MAPK under lipopolysaccharide stimulation and down-regulated Tnfα, and Ttp mRNA. In addition, hyper-phosphorylation of Zfp36l1 might stabilize Mkp-1 expression by forming a complex with the adapter protein 14-3-3 and decreasing the interaction with deadenylase Caf1a.ConclusionsOur findings imply that the expression and phosphorylation of Zfp36l1 and Zfp36l2 may modulate the basal level of Mkp-1 mRNA to control p38 MAPK activity during lipopolysaccharide stimulation, which would affect the inflammatory mediators production. Zfp36l1 and Zfp36l2 are important regulators of the innate immune response.Electronic supplementary materialThe online version of this article (doi:10.1186/s12950-015-0088-x) contains supplementary material, which is available to authorized users.

Background Tristetraprolin binds mRNA AU-rich elements and thereby facilitates the destabilization of mature mRNA in the cytosol. Methodology/Principal Findings To understand how tristetraprolin mechanistically functions, we biopanned with a phage-display library for proteins that interact with tristetraprolin and retrieved, among others, a fragment of poly(A)-binding protein nuclear 1, which assists in the 3'-polyadenylation of mRNA by binding to immature poly(A) tails and thereby increases the activity of poly(A) polymerase, which is directly responsible for polyadenylation. The tristetraprolin/poly(A)-binding protein nuclear 1 interaction was characterized using tristetraprolin and poly(A)-binding protein nuclear 1 deletion mutants in pull-down and co-immunoprecipitation assays. Tristetraprolin interacted with the carboxyl-terminal region of poly(A)-binding protein nuclear 1 via its tandem zinc finger domain and another region. Although tristetraprolin and poly(A)-binding protein nuclear 1 are located in both the cytoplasm and the nucleus, they interacted in vivo in only the nucleus. In vitro , tristetraprolin bound both poly(A)-binding protein nuclear 1 and poly(A) polymerase and thereby inhibited polyadenylation of AU-rich element–containing mRNAs encoding tumor necrosis factor α, GM-CSF, and interleukin-10. A tandem zinc finger domain–deleted tristetraprolin mutant was a less effective inhibitor. Expression of a tristetraprolin mutant restricted to the nucleus resulted in downregulation of an AU-rich element–containing tumor necrosis factor α/luciferase mRNA construct. Conclusion/Significance In addition to its known cytosolic mRNA–degrading function, tristetraprolin inhibits poly(A) tail synthesis by interacting with poly(A)-binding protein nuclear 1 in the nucleus to regulate expression of AU-rich element–containing mRNA.

The Tristetraprolin (TTP) protein family includes four mammalian members (TTP, TIS11b, TIS11d, and ZFP36L3), but only one in Drosophila melanogaster (DTIS11). These proteins bind target mRNAs with AU-rich elements (AREs) via two C3H zinc finger domains and destabilize the mRNAs. We found that overexpression of mouse TIS11b or DTIS11 in the Drosophila retina dramatically reduced eye size, similar to the phenotype of eyes absent (eya) mutants. The eya transcript is one of many ARE-containing mRNAs in Drosophila. We showed that TIS11b reduced levels of eya mRNA in vivo. In addition, overexpression of Eya rescued the TIS11b overexpression phenotype. RNA pull-down and luciferase reporter analyses demonstrated that the DTIS11 RNA-binding domain is required for DTIS11 to bind the eya 3′ UTR and reduce levels of eya mRNA. Moreover, ectopic expression of DTIS11 in Drosophila S2 cells decreased levels of eya mRNA and reduced cell viability. Consistent with these results, TTP proteins overexpressed in MCF7 human breast cancer cells were associated with eya homologue 2 (EYA2) mRNA, and caused a decrease in EYA2 mRNA stability and cell viability. Our results suggest that eya mRNA is a target of TTP proteins, and that downregulation of EYA by TTP may lead to reduced cell viability in Drosophila and human cells.

A new class of metallo-hydrogels has been developed using di(2-picolyl)amine (DPA)-functionalized 4-arm polyethylene glycol (4A-PEG-DPAn) polymers crosslinked by metal–ligand coordination.

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Endothelial NO synthase (eNOS) function is critically modulated by protein phosphorylation. In particular, phosphorylation of serine 1179 (S1179, bovine)/1177 (S1177, human and rat) by Akt has emerged as a central mechanism of eNOS regulation under both physiological and pathological conditions. Endoplasmic reticulum (ER) stress is a fundamental unfolded protein response occurred in various diseases. Whether and how ER stress affects eNOS phosphorylation is unknown. To address this issue, we induced ER stress in bovine aortic endothelial cells (BAECs) with Brefeldin A (BFA, 5 μg/ml), a compound blocking protein transport from ER to Golgi apparatus. BFA time-dependently induced ER stress in BAECs as evidenced by the markedly increased expressions of ER chaperon Grp78. Parallel to the time course of ER stress, a progressive loss of eNOS S1179 phosphorylation was seen. ER stress-induced eNOS dephosphorylation was specific to S1179 because the phosphorylation status of eNOS T497 or S635 was unchanged. In cells exposed to BFA for 4 hr, eNOS S1179 phosphorylation was decreased more than 5 fold (17.9±0.1% of control, P <0.01, n=5). As a result, eNOS activity was diminished (from 3.32±0.28 to 0.85±0.08 pmol/mg/min, P <0.01, n=3). Further studies revealed that ER stress caused Akt T308 and S473 dephosphorylation leading to Akt deactivation. Besides BFA, the loss of eNOS and Akt phosphorylation was also measured in ER stress induced by depleting ER Ca 2+ content with A23187 (2 μM) or perturbing ER oxidative environment with DTT (5 mM). To determine if these findings from cell culture occur in vivo, we monitored ER stress and eNOS S1177 phosphorylation in postischemic rat hearts. Indeed, severe ER stress and corresponding loss of eNOS S1177 phosphorylation and activity were detected in the infarcted areas of hearts after 1-hr coronary artery (LAD) occlusion followed by 24-hr reperfusion. Collectively, these results demonstrate that ER stress decreases eNOS S1179 phosphorylation and function via Akt deactivation. Ischemia/reperfusion cause ER stress, which, at least in part, accounts for the loss of eNOS S1179 phosphorylation and function in hearts. Thus, reducing ER stress may be an important approach to prevent eNOS dysfunction in postischemic hearts.