CNOT10 is involved in the TTP-mediated AU-rich element-containing mRNA metabolism, independent of mRNA decay regulation.

Tristetraprolin (TTP)/ZFP36 is an RNA binding protein that is involved in the turnover regulation of the target adenylate-uridylate-rich RNA element (ARE)-containing mRNAs. AREs are present in the 3'-untranslated region of transcripts expressed from many immediate-early genes, including cytokines and chemokines. It has been demonstrated that TTP-mediated posttranscriptional mRNA decay regulation is crucial for modulating physiological control, particularly in response to inflammatory stimulation. TTP is associated with the CCR4-NOT deadenylation complex through the TTP C-terminus to promote mRNA decay. However, it is not fully understood whether there are additional sites within TTP that contribute to its function through interacting with other factors. We analyzed the functionality of the unique tryptophan residues located in the TTP N-terminus using a cell-based assay system that consists of a tetracycline-responsive CMV promoter-driven, intron-inserted luciferase (LUC). This system enabled us to analyze TTP activity during both the early phase and the steady-state phase of gene expression, as well as in the posttranscriptional mRNA decay following the treatment of tetracycline analogs. Meanwhile, we identified putative TTP associates using a proximity labeling method. We found that tryptophan residues in the TTP N-terminus together with CNOT10, a component of the CCR4-NOT complex, were involved specifically in the reduction of the ARE-containing LUC mRNA level during the early phase of gene expression. However, they were not involved in the decay of LUC mRNA in the steady-state phase. We propose a novel posttranscriptional TTP functionality in the reduction of ARE-containing mRNA levels, which differs from the well-characterized mRNA decay activity.