The inflammasome regulates release of caspase activation-dependent cytokines, including IL-1β, IL-18, and high-mobility group box 1 (HMGB1)1-5. During the course of studying HMGB1 release mechanisms, we discovered an important role of double-stranded RNA dependent protein kinase (PKR) in inflammasome activation. Exposure of macrophages to inflammasome agonists induced PKR autophosphorylation. PKR inactivation by genetic deletion or pharmacological inhibition severely impaired inflammasome activation in response to double-stranded RNA, ATP, monosodium urate, adjuvant aluminum, rotenone, live E. coli, anthrax lethal toxin, DNA transfection, and S. Typhimurium infection. PKR deficiency significantly inhibited the secretion of IL-1beta, IL-18 and HMGB1 in E. coli-induced peritonitis. PKR physically interacts with multiple inflammasome components, including NLR family pyrin domain-containing 3 (NLRP3), NLR family pyrin domain-containing 1 (NLRP1), NLR family CARD domain-containing protein 4 (NLRC4), Absent in melanoma 2 (AIM2), and broadly regulates inflammasome activation. PKR autophosphorylation in a cell free system with recombinant NLRP3, ASC and pro-casapse-1 reconstitutes inflammasome activity. These results reveal a critical role of PKR in inflammasome activation, and indicate that it should be possible to pharmacologically target this molecule to treat inflammation.

SUMMARY As chronic inflammation is a hallmark of obesity, pathways that integrate nutrient and pathogen sensing pathways are of great interest in understanding the mechanisms of insulin resistance, type 2 diabetes, and other chronic metabolic pathologies. Here, we provide evidence that double-stranded RNA dependent protein kinase (PKR) can respond to nutrient signals as well as endoplasmic reticulum (ER) stress and coordinate the activity of other critical inflammatory kinases such as the c-Jun N-terminal kinase (JNK) to regulate insulin action and metabolism. PKR also directly targets and modifies insulin receptor substrate and hence integrates nutrients and insulin action with a defined pathogen response system. Dietary and genetic obesity features marked activation of PKR in adipose and liver tissues and absence of PKR alleviates metabolic deterioration due to nutrient or energy excess in mice. These findings demonstrate PKR as a critical component of an inflammatory complex that responds to nutrients and organelle dysfunction.

tob is a member of an emerging family of genes with antiproliferative function. Tob is rapidly phosphorylated at Ser 152, Ser 154, and Ser 164 by Erk1 and Erk2 upon growth-factor stimulation. Oncogenic Ras-induced transformation and growth-factor-induced cell proliferation are efficiently suppressed by mutant Tob that carries alanines but not glutamates, mimicking phosphoserines, at these sites. Wild-type Tob inhibits cell growth when the three serine residues are not phosphorylated but is less inhibitory when the serines are phosphorylated. Because growth of Rb-deficient cells was not affected by Tob, Tob appears to function upstream of Rb. Intriguingly, cyclin D1 expression is elevated in serum-starved tob −/− cells. Reintroduction of wild-type Tob and mutant Tob with serine-to-alanine but not to glutamate mutations on the Erk phosphorylation sites in these cells restores the suppression of cyclin D1 expression. Finally, the S-phase population was significantly increased in serum-starved tob −/− cells as compared with that in wild-type cells. Thus, Tob inhibits cell growth by suppressing cyclin D1 expression, which is canceled by Erk1-and Erk2-mediated Tob phosphorylation. We propose that Tob is critically involved in the control of early G 1 progression.

The stability of mRNA influences the abundance of cellular transcripts and proteins. Deadenylases play critical roles in mRNA turnover and thus are important for the regulation of various biological events. Here, we report the identification and characterization of CCR4b/CNOT6L, which is homologous to yeast CCR4 mRNA deadenylase. CCR4b is localized mainly in the cytoplasm and displays deadenylase activity both in vitro and in vivo. CCR4b forms a multisubunit complex similar to the yeast CCR4-NOT complex. Suppression of CCR4b by RNA interference results in growth retardation of NIH 3T3 cells accompanied by elevation of both p27Kip1 mRNA and p27 Kip1 protein. Reintroduction of wild-type CCR4b, but not mutant CCR4b lacking deadenylase activity, restores the growth of CCR4b-depleted NIH 3T3 cells. The data suggest that CCR4b regulates cell growth in a manner dependent on its deadenylase activity. We also show that p27Kip1 mRNA is stabilized and its poly(A) tail is preserved in CCR4b-depleted cells. Our findings provide evidence that CCR4b deadenylase is a constituent of the mammalian CCR4-NOT complex and regulates the turnover rate of specific target mRNAs. Thus, CCR4b may be involved in various cellular events that include cell proliferation.Regulation of mRNA turnover rates is important in determining the abundance and translational efficiencies of mRNAs. Removal of the 3Ј poly(A) tail triggers degradation of mRNAs. Two mechanisms have been described for the completion of mRNA degradation. With the first mechanism, a decapping complex consisting of Dcp1 and Dcp2 recognizes the deadenylated mRNA and hydrolyzes the m 7 GpppN cap (5Ј capping structure of mRNA), leading to 5Ј-to-3Ј exonuclease digestion catalyzed by Xrn1. In the other mechanism, deadenylation is followed by 3Ј-to-5Ј degradation of the deadenylated mRNA by the exosome complex containing 3Ј-to-5Ј exonuclease. In either case, deadenylation is a critical step in mRNA degradation and regulates the abundance of mRNA (for reviews, see references 22 and 34).Each mRNA has a distinct degradation rate. Although the precise mechanism of deadenylation remains to be established, the deadenylation rate of each mRNA would influence the mRNA degradation rate. Sequence elements that are responsible, at least in part, for mRNA turnover by promoting deadenylation have been identified in the c-Fos, interleukin 2, and tumor necrosis factor alpha genes (5, 12, 15). One of the best-studied and most prevalent elements is the AU-rich element (ARE), which is found in the 3Ј untranslated region (3Ј UTR) of mRNAs. AREs in the interleukin 2 mRNA and tumor necrosis factor alpha mRNA promote rapid deadenylation-dependent mRNA decay (5, 15). In addition, because the 3Ј poly(A) tail can enhance translation, deadenylation influences the efficiency of translation (4, 23). Thus, deadenylases are crucial for gene expression and therefore are likely to be important for many biological processes.Two distinct enzyme complexes, Pan2-Pan3 and CCR4-NOT, have been identified in yeast as mRNA dea...

The Tob/BTG family is a group of antiproliferative proteins containing two highly homologous regions, Box A and Box B. These proteins all associate with CCR4-associated factor 1 (Caf1), which belongs to the ribonuclease D (RNase D) family of deadenylases and is a component of the CCR4-Not deadenylase complex. Here we determined the crystal structure of the complex of the N-terminal region of Tob and human Caf1 (hCaf1). Tob exhibited a novel fold, whereas hCaf1 most closely resembled the catalytic domain of yeast Pop2 and human poly(A)-specific ribonuclease. Interestingly, the association of hCaf1 was mediated by both Box A and Box B of Tob. Cell growth assays using both wild-type and mutant proteins revealed that deadenylase activity of Caf1 is not critical but complex formation is crucial to cell growth inhibition. Caf1 tethers Tob to the CCR4-Not deadenylase complex, and thereby Tob gathers several factors at its C-terminal region, such as poly(A)-binding proteins, to exert antiproliferative activity.

Spermatogenesis is a complex process that involves cooperation of germ cells and testicular somatic cells. Various genetic disorders lead to impaired spermatogenesis, defective sperm function and male infertility 1 . Here we show that Cnot7 -/-males are sterile owing to oligo-astheno-teratozoospermia, suggesting that Cnot7, a CCR4-associated transcriptional cofactor 2 , is essential for spermatogenesis. Maturation of spermatids is unsynchronized and impaired in seminiferous tubules of Cnot7 -/-mice. Transplantation of spermatogonial stem cells from male Cnot7 -/-mice to seminiferous tubules of Kit mutant mice (Kit W/W-v ) restores spermatogenesis, suggesting that the function of testicular somatic cells is damaged in the Cnot7 -/-condition. The testicular phenotypes of Cnot7 -/-mice are similar to those of mice deficient in retinoid X receptor beta (Rxrb) 3 . We further show that Cnot7 binds the AF-1 domain of Rxrb and that Rxrb malfunctions in the absence of Cnot7. Therefore, Cnot7 seems to function as a coregulator of Rxrb in testicular somatic cells and is thus involved in spermatogenesis.

Protein kinase RNA-activated (PKR) has long been known to be activated by viral double-stranded RNA (dsRNA) as part of the mammalian immune response. However, in mice PKR is also activated by metabolic stress in the absence of viral infection, and this requires a functional kinase domain, as well as a functional dsRNA-binding domain. The endogenous cellular RNA that potentially leads to PKR activation during metabolic stress is unknown. We investigated this question using mouse embryonic fibroblast cells expressing wild-type PKR (PKR WT ) or PKR with a point mutation in each dsRNA-binding motif (PKR RM ). Using this system, we identified endogenous RNA that interacts with PKR after induction of metabolic stress by palmitic acid (PA) treatment. Specifically, RIP-Seq analyses showed that the majority of enriched RNAs that interacted with WT PKR (≥twofold, false discovery rate ≤ 5%) were small nucleolar RNAs (snoRNAs). Immunoprecipitation of PKR in extracts of UV-cross-linked cells, followed by RT-qPCR, confirmed that snoRNAs were enriched in PKR WT samples after PA treatment, but not in the PKR RM samples. We also demonstrated that a subset of identified snoRNAs bind and activate PKR in vitro; the presence of a 5′-triphosphate enhanced PKR activity compared with the activity with a 5′-monophosphate, for some, but not all, snoRNAs. Finally, we demonstrated PKR activation in cells upon snoRNA transfection, supporting our hypothesis that endogenous snoRNAs can activate PKR. Our results suggest an unprecedented and unexpected model whereby snoRNAs play a role in the activation of PKR under metabolic stress.PKR | snoRNA | metabolic stress | phosphorylation | RNA-binding protein

tob is a member of antiproliferative family genes. Mice lacking tob are prone to spontaneous formation of tumors. The occurrence rate of diethylnitrosamine-induced liver tumors is higher in tob −/− mice than in wildtype mice. tob −/− p53 −/− mice show accelerated tumor formation in comparison with single null mice. Expression of cyclin D1 mRNA is increased in the absence of Tob and is reduced by Tob. Tob acts as a transcriptional corepressor and suppresses the cyclin D1 promoter activity through an interaction with histone deacetylase. Levels of tob mRNA are often decreased in human cancers, implicating tob in cancer development.Supplemental material is available at http://www.genesdev.org. There is accumulating evidence that genes involved in the negative control of cell growth can function as tumor suppressors. In humans, tob, tob2, ana, pc3b, btg1, and btg2 comprise a family (tob family) of antiproliferative genes (Bradbury et al. 1991;Fletcher et al. 1991;Rouault et al. 1992;Matsuda et al. 1996;Guehenneux et al. 1997;Yoshida et al. 1998;Ikematsu et al. 1999;Buanne et al. 2000). Exogenous expression of Tob family proteins suppresses growth of NIH-3T3 cells by inhibiting G1 progression of the cell cycle (Yoshida et al. 1998;Ikematsu et al. 1999;Guardavaccaro et al. 2000;Maekawa et al. 2002;Suzuki et al. 2002). We showed previously that Tob is a substrate of Erk MAPK, and unphosphorylated Tob suppresses cell-cycle entry of quiescent cells. Erk phosphorylation of Tob blocks the antiproliferative activity (Maekawa et al. 2002;Suzuki et al. 2002), which, at least in part, describes the importance of Erk activation in the cells stimulated by growth factors. When Tob is depleted, Cyclin D1 continues to be expressed and readily progress into S phase during serum starvation (Suzuki et al. 2002). In addition, the antiproliferative activity of Tob is impaired in the presence of exogenously coexpressed Cyclin D1 (Suzuki et al. 2002). These data suggest that tob functions as a tumor suppressor. However, possible involvement of Tob in tumorigenesis and roles of Tob in the control of cyclin D1 expression are unclear.Tob family proteins associate with transcription factors. Virtually all of the Tob family members interact with Caf1 (Rouault et al. 1998;Ikematsu et al. 1999;Yoshida et al. 2001), whose yeast homolog is a component of the CCR4-NOT transcriptional complex (Albert et al. 2000). The CCR4-NOT complex participates in the control of specific sets of genes such as those involved in the late mitotic phase of the cell cycle (Liu et al. 1997). Both BTG1 and BTG2 associate with HoxB9 and estrogen receptor ␣, and modulate their transcription activity (Prevot et al. 2000(Prevot et al. , 2001. Tob associates with Smads transcription complex and affects Smad-mediated gene expression (Yoshida et al. 2000;Tzachanis et al. 2001). This suggests that Tob family proteins are regulators of gene transcription, functioning as either coactivators or corepressors.Here, we report that mice lacking tob are prone to spontaneous formation o...