The Drosophila TEAD ortholog Scalloped is required for Yki-mediated overgrowth but is largely dispensable for normal tissue growth, suggesting that its mammalian counterpart may be exploited for selective inhibition of oncogenic growth driven by YAP hyperactivation. Here we test this hypothesis genetically and pharmacologically. We show that a dominant-negative TEAD molecule does not perturb normal liver growth but potently suppresses hepatomegaly/tumorigenesis resulting from YAP overexpression or Neurofibromin 2 (NF2)/Merlin inactivation. We further identify verteporfin as a small molecule that inhibits TEAD-YAP association and YAPinduced liver overgrowth. These findings provide proof of principle that inhibiting TEAD-YAP interactions is a pharmacologically viable strategy against the YAP oncoprotein.

Elongator has been reported to be a histone acetyltransferase complex involved in elongation of RNA polymerase II transcription. In Saccharomyces cerevisiae, mutations in any of the six Elongator protein subunit (ELP1-ELP6) genes or the three killer toxin insensitivity (KTI11-KTI13) genes cause similar pleiotropic phenotypes. By analyzing modified nucleosides in individual tRNA species, we show that the ELP1-ELP6 and KTI11-KTI13 genes are all required for an early step in synthesis of 5-methoxycarbonylmethyl (mcm 5 ) and 5-carbamoylmethyl (ncm 5 ) groups present on uridines at the wobble position in tRNA. Transfer RNA immunoprecipitation experiments showed that the Elp1 and Elp3 proteins specifically coprecipitate a tRNA susceptible to formation of an mcm 5 side chain, indicating a direct role of Elongator in tRNA modification. The presence of mcm 5 U, ncm 5 U, or derivatives thereof at the wobble position is required for accurate and efficient translation, suggesting that the phenotypes of elp1-elp6 and kti11-kti13 mutants could be caused by a translational defect. Accordingly, a deletion of any ELP1-ELP6 or KTI11-KTI13 gene prevents an ochre suppressor tRNA that normally contains mcm 5 U from reading ochre stop codons.

The Saccharomyces cerevisiae Elongator complex consisting of the six Elp1-Elp6 proteins has been proposed to participate in three distinct cellular processes: transcriptional elongation, polarized exocytosis, and formation of modified wobble uridines in tRNA. Therefore it was important to clarify whether Elongator has three distinct functions or whether it regulates one key process that leads to multiple downstream effects. Here, we show that the phenotypes of Elongator-deficient cells linking the complex to transcription and exocytosis are suppressed by increased expression of two tRNA species. Elongator is required for formation of the mcm(5) group of the modified wobble nucleoside 5-methoxycarbonylmethyl-2-thiouridine (mcm(5)s(2)U) in these tRNAs. Hence, in cells with normal levels of these tRNAs, presence of mcm(5)s(2)U is crucial for posttranscriptional expression of gene products important in transcription and exocytosis. Our results indicate that the physiologically relevant function of the evolutionary-conserved Elongator complex is in formation of modified nucleosides in tRNAs.

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Kluyveromyces lactis killer strains secrete a heterotrimeric toxin (zymocin), which causes an irreversible growth arrest of sensitive yeast cells. Despite many efforts, the target(s) of the cytotoxic g-subunit of zymocin has remained elusive. Here we show that three tRNA species tRNA . Transfer RNA lacking a part of or the entire mcm 5 group is inefficiently cleaved by g-toxin, explaining the g-toxin resistance of the modification-deficient trm9, elp1-elp6, and kti11-kti13 mutants. The K. lactis g-toxin is the first eukaryotic toxin shown to target tRNA.

The Hippo tumor suppressor pathway restricts tissue growth by inactivating the transcriptional coactivator Yki. Although Sd has been implicated as a DNA-binding transcription factor partner for Yki and can genetically account for gain-of-function Yki phenotypes, how Yki regulates normal tissue growth remains a long-standing puzzle since Sd, unlike Yki, is dispensable for normal growth in most Drosophila tissues. Here we show that the yki mutant phenotypes in multiple developmental contexts are rescued by inactivation of Sd, suggesting that Sd functions as a default repressor and that Yki promotes normal tissue growth by relieving Sd-mediated default repression. We further identify Tgi as a cofactor involved in Sd's default repressor function and demonstrate that the mammalian orthologue of Tgi potently suppresses the YAP oncoprotein in transgenic mice. These findings fill a major gap in Hippo-mediated transcriptional regulation and open up new possibilities for modulating the YAP oncoprotein in cancer and regenerative medicine.

Inflammasomes are multi-protein complexes that trigger the activation of caspase-1 and the maturation of interleukin-1β (IL-1β), yet the regulation of these complexes remains poorly characterized. Here we show that nitric oxide (NO) inhibited the NLRP3-mediated ASC pyroptosome formation, caspase-1 activation and IL-1β secretion in myeloid cells from both mice and humans. Meanwhile, endogenous NO derived from iNOS (inducible form of NO synthase) also negatively regulated NLRP3 inflammasome activation. Depletion of iNOS resulted in increased accumulation of dysfunctional mitochondria in response to LPS and ATP, which was responsible for the increased IL-1β production and caspase-1 activation. iNOS deficiency or pharmacological inhibition of NO production enhanced NLRP3-dependent cytokine production in vivo, thus increasing mortality from LPS-induced sepsis in mice, which was prevented by NLRP3 deficiency. Our results thus identify NO as a critical negative regulator of the NLRP3 inflammasome via the stabilization of mitochondria. This study has important implications for the design of new strategies to control NLRP3-related diseases.

Transfer RNAs specific for Gln, Lys, and Glu from all organisms (except Mycoplasma) and organelles have a 2-thiouridine derivative (xm 5 s 2 U) as wobble nucleoside. These tRNAs read the A-and G-ending codons in the split codon boxes His/Gln, Asn/Lys, and Asp/Glu. In eukaryotic cytoplasmic tRNAs the conserved constituent (xm 5 -) in position 5 of uridine is 5-methoxycarbonylmethyl (mcm 5 ). A protein (Tuc1p) from yeast resembling the bacterial protein TtcA, which is required for the synthesis of 2-thiocytidine in position 32 of the tRNA, was shown instead to be required for the synthesis of 2-thiouridine in the wobble position (position 34). Apparently, an ancient member of the TtcA family has evolved to thiolate U34 in tRNAs of organisms from the domains Eukarya and Archaea. Deletion of the TUC1 gene together with a deletion of the ELP3 gene, which results in the lack of the mcm 5 side chain, removes all modifications from the wobble uridine derivatives of the cytoplasmic tRNAs specific for Gln, Lys, and Glu, and is lethal to the cell. Since excess of the unmodified form of these three tRNAs rescued the double mutant elp3 tuc1, the primary function of mcm 5 s 2 U34 seems to be to improve the efficiency to read the cognate codons rather than to prevent mis-sense errors. Surprisingly, overexpression of the mcm 5 s 2 U-lacking tRNA Lys alone was sufficient to restore viability of the double mutant.