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Tristetraprolin (TTP) is the prototype for a family of RNA binding proteins that bind the tumor necrosis factor (TNF) messenger RNA AU-rich element (ARE), causing deadenylation of the TNF poly(A) tail, RNA decay, and silencing of TNF protein production. Using mass spectrometry sequencing we identified poly(A) binding proteins-1 and -4 (PABP1 and PABP4) in high abundance and good protein coverage from TTP immunoprecipirates. PABP1 significantly enhanced TNF ARE binding by RNA EMSA and prevented TTP-initiated deadenylation in an in vitro macrophage assay of TNF poly(A) stability. Neomycin inhibited TTP-promoted deadenylation at concentrations shown to inhibit the deadenylases poly(A) ribonuclease and CCR4. Stably transfected RAW264.7 macrophages overexpressing PABP1 do not oversecrete TNF; instead they upregulate TTP protein without increasing TNF protein production. The PABP1 inhibition of deadenylation initiated by TTP does not require the poly(A) binding regions in RRM1 and RRM2, suggesting a more complicated interaction than simple masking of the poly(A) tail from a 3'-exonuclease. Like TTP, PABP1 is a substrate for p38 MAP kinase. Finally, PABP1 stabilizes cotransfected TTP in 293T cells and prevents the decrease in TTP levels seen with p38 MAP kinase inhibition. These findings suggest several levels of functional antagonism between TTP and PABP1 that have implications for regulation of unstable mRNAs like TNF. RNA; immunology; tumor necrosis factor

A genome study is presented with relevance to the ecologically successful protozoa Tetrahymena.

Wnt signaling acts in part through the low density lipoprotein receptor-related transmembrane proteins LRP5 and LRP6 to regulate embryonic development and stem cell proliferation. Up-regulated signaling is associated with many forms of cancer. Casein kinase I e (CKIϵ) is a known component of the Wnt-β-catenin signaling pathway. We find that CKIϵ binds to LRP5 and LRP6 in vitro and in vivo and identify three CKIϵ-specific phosphorylation sites in LRP6. Two of the identified phosphorylation sites, Ser142° and Ser'430, influence Wnt signaling in vivo, since LRP6 with mutation of these sites is a more potent activator of both β-catenin accumulation and Lef-1 reporter activity. Whereas Wnt3a regulates CKIϵ kinase activity, LRP6 does not, placing CKIϵ upstream of LRP6. Mutation of LRP6 Ser142° and Ser'43° to alanine strengthens its interaction with axin, suggesting a mechanism by which CKIϵ may negatively regulate Wnt signaling. The role of CKIϵ is therefore more complex than was previously appreciated. Generation of active CKIϵ may induce a negative feedback loop by phosphorylation of sites on LRP5/6 that modulate axin binding and hence β-catenin degradation. [ABSTRACT FROM AUTHOR]