Our results show that upon TLR4 activation, TUT7, but not TUT4, is upregulated and TUT7 subsequently associates with and oligouridylates to regulate cytokine production. Here we reveal that TLR4 activation induces TUT7, which in turn selectively regulates the production of a subset of cytokines, including Interleukin 6 (IL-6). TUT7 regulates IL-6 expression by controlling ribonuclease Regnase-1 mRNA (encoded by gene) stability. Mechanistically, TLR4 activation causes TUT7 to bind directly to the stem-loop structure on 3-UTR, thereby promotes uridylation and degradation. from LPS-treated TUT7-sufficient macrophages possesses increased oligo-uridylated ends with shorter poly(A) tails, whereas oligo-uridylated is usually significantly reduced in gene) modulates IL-6 mRNA stability when macrophages are activated by LPS or IL-115. Mechanistic studies revealed that Regnase-1 targets the stem-loop structure on 3-UTR and cleaves mRNA through cooperation with RNA helicase protein, UPF111,16. On the other hand, activation with LPS and cytokines also enhances the expression of Arid5a. The presence of Arid5a impedes Regnase-1 binding to 3-UTR thereby stabilizes mRNA12. Therefore, it appears that regulation of spatiotemporal expression of RBPs can fine tune the quantity of cytokine mRNAs during inflammation. Recent studies show that modification of RNAs on their 3 ends by non-templated nucleotide addition is an evolutionary conserved mechanism Ecscr for the control of RNA stability and its fate17. Among the known template-independent RNA 3 terminal modifications, polyadenylation and uridylation are the two most analyzed mechanisms18. Uridylation is usually catalyzed by terminal uridyltransferases (TUTs). TUTs belong to a family of non-canonical poly(A) polymerases. Their function in RNA processing is usually evolutionally conserved from LY2119620 to human19. TUT4 (also known as ZCCHC11) and TUT7 (also named ZCCHC6) are primarily responsible for cytoplasmic 3 uridylation20,21 of various RNAs, including precursor microRNAs (pre-miRNAs)22,23, mature miRNAs24, histone mRNAs25,26, cellular mRNAs20, noncoding RNAs27, and viral RNAs28 in mammalian cells. Catalytic activities of TUT4/7 can mono-uridylate or oligo-uridylate pre-miRNA resulting in biogenesis or degradation of miRNAs, respectively21,23. TUT4/7 are also known to trigger oligo-uridylation of LY2119620 poly(A)-tail-lacking histone mRNA at the end of S phase of a cell cycle to enhance its degradation25,26,29. Recently, TAIL-seq analysis revealed that TUT4 and TUT7 uridylate mRNAs with short poly(A) tails, leading to 5-to-3 or 3-to-5 mRNA degradation by recruiting deadenylases, decapping enzymes, and exonucleases20,30,31. These studies show that oligo-uridylation is usually associated with RNA degradation, but how TUT4/7 uridylate RNAs remains unknown. TUTs are recently shown to modulate inflammatory responses in mammalian cells. TUT4 uridylates and degrades IL-6-targeting miR-26a/b, leading to control of the quantity of mRNA in A549 cells following TNF treatment24. TUT7 is also known to modulate miR-26b uridylation and stability in IL-1-stimulated chondrocytes through which positively regulates IL-6 expression32. A recent statement revealed that TUT7 impedes the expression of a specific set of pro-inflammatory cytokines (including IL-6) in mice after challenge with mRNA decay by uridylating its 3 end, which subsequently prevents Regnase-1 from degrading isolated from LPS-treated TUT7-sufficient bone marrow-derived macrophages (BMDMs) contains increased oligo-uridines (2?U) with short poly(A) tails at its 3 end, whereas reduced oligo-uridylation is found in transcripts from TUT7-deficient cells after the same treatment. Together, our results demonstrate that TUT7 functions as LY2119620 a regulator in TLR4-driven inflammatory responses by mediating uridylation of and thus destabilizing the mRNAs of inflammatory mediators including and mRNA expression was determined by RT-qPCR (b, d). The levels of indicated inflammatory cytokines in cultural media were analyzed by ELISA (c, d). The levels of pro-IL-1, LY2119620 IB, and phosphorylation of MAP kinases were analyzed by immunoblotting (e, f). g Volcano plot of the changes of innate immune-related gene expressions in values to the base 2 of the fold-change (FC) and the value?=?0.05). log2FC? ?+0.2 and log2FC? ??0.2 indicate increase of transcript levels by 20% and decreased by 20%, respectively. Green and red dots denote transcripts related to innate immune responses that were significantly lower (values were obtained from two-tailed Students test and are shown in the figure if in RAW 264.7 macrophages significantly decreased mRNA, and cytosolic pro-IL-1, and IL-6 and IL-12p40, but not and mRNA or TNF protein expression upon LPS challenge (Supplementary Fig.?1dCh). Nevertheless, LPS-induced IB degradation, activation of MAPKs JNK and p38, and nuclear translocation of IRF3 were comparable in control and TUT7-silenced cells (Supplementary Fig.?1i, j). These results suggest a regulatory role for TUT7 in TLR4-mediated immune response. We.