Utp9p is a nucleolar protein that is part of a subcomplex containing several U3 snoRNA-associated proteins including Utp8p, which is a protein that shuttles aminoacyl-tRNAs from the nucleolus to the nuclear tRNA export receptors Los1p and Msn5p in Saccharomyces cerevisiae. Here we show that Utp9p is also an intranuclear component of the Msn5p-mediated nuclear tRNA export pathway. Depletion of Utp9p caused nuclear accumulation of mature tRNAs derived from intron-containing precursors, but not tRNAs made from intronless pre-tRNAs. Utp9p binds tRNA directly and saturably, and copurifies with Utp8p, Gsp1p, and Msn5p, but not with Los1p or aminoacyl-tRNA synthetases. Utp9p interacts directly with Utp8p, Gsp1p, and Msn5p in vitro. Furthermore, Gsp1p forms a complex with Msn5p and Utp9p in a tRNA-dependent manner. However, Utp9p does not shuttle between the nucleus and the cytoplasm. Because tRNA splicing occurs in the cytoplasm and the spliced tRNAs are retrograded back to the nucleus, we propose that Utp9p facilitates nuclear reexport of retrograded tRNAs. Moreover, the data suggest that Utp9p together with Utp8p translocate aminoacyl-tRNAs from the nucleolus to Msn5p and assist with formation of the Msn5p-tRNA-Gsp1p-GTP export complex.

BackgroundSTAT1 and IRF1 collaborate to induce interferon-γ (IFNγ) stimulated genes (ISGs), but the extent to which they act alone or together is unclear. The effect of single nucleotide polymorphisms (SNPs) on in vivo binding is also largely unknown.ResultsWe show that IRF1 binds at proximal or distant ISG sites twice as often as STAT1, increasing to sixfold at the MHC class I locus. STAT1 almost always bound with IRF1, while most IRF1 binding events were isolated. Dual binding sites at remote or proximal enhancers distinguished ISGs that were responsive to IFNγ versus cell-specific resistant ISGs, which showed fewer and mainly single binding events. Surprisingly, inducibility in one cell type predicted ISG-responsiveness in other cells. Several dbSNPs overlapped with STAT1 and IRF1 binding motifs, and we developed methodology to rapidly assess their effects. We show that in silico prediction of SNP effects accurately reflects altered binding both in vitro and in vivo.ConclusionsThese data reveal broad cooperation between STAT1 and IRF1, explain cell type specific differences in ISG-responsiveness, and identify genetic variants that may participate in the pathogenesis of immune disorders.Electronic supplementary materialThe online version of this article (doi:10.1186/s12867-017-0084-1) contains supplementary material, which is available to authorized users.

In Saccharomyces cerevisiae intron-containing pre-tRNAs are exported from the nucleus to the cytoplasm for removal of the introns, and the spliced tRNAs are returned to the nucleus for reasons that are not understood. The re-imported spliced tRNAs are then subjected to aminoacylation in the nucleolus to ensure that they are functional prior to re-export to the cytoplasm. Previous studies have shown that re-imported spliced tRNAs and mature tRNAs made entirely in the nucleus from intronless precursors are retained in the nucleus of S. cerevisiae in response to glucose, amino acid, nitrogen or inorganic phosphate deprivation. Contrary to these studies, we recently reported that starvation of S. cerevisiae of amino acids or nitrogen results in nuclear accumulation of re-imported spliced tRNAs, but not tRNAs made from intronless precursors. This finding suggests that separate pathways are used for nuclear export of retrogradely transported spliced tRNAs and tRNAs made from intronless pre-tRNAs. In addition, the data support the conclusion that the nuclear re-export pathway for retrogradely transported spliced tRNAs, but not the pathway responsible for nuclear export of tRNAs derived from intronless precursors is regulated during amino acid or nitrogen starvation. This regulation appears to occur at a step after the re-imported spliced tRNAs have undergone aminoacylation quality assurance and, in part, involves the TORC1 signalling pathway. Moreover, it was established that Utp9p is an intranuclear component that only facilitates nuclear re-export of retrogradely transported spliced tRNAs by the β-karyopherin Msn5p. Utp9p acts in concert with Utp8p, a key player in nuclear tRNA export in S. cerevisiae, to translocate aminoacylated re-imported spliced tRNAs from the nucleolus to Msn5p and assist with formation of the Msn5p-tRNA-Gsp1p-GTP export complex. This pathway, however, is not the only one responsible for nuclear re-export of retrogradely transported spliced tRNAs.

Polycomb Repressive Complex 2 (PRC2) is an epigenetic regulator induced in many cancers. It is thought to drive tumorigenesis by repressing division, stemness, and/or developmental regulators. Cancers evade immune detection, and diverse immune regulators are perturbed in different tumors. It is unclear how such cell-specific effects are coordinated. Here, we show a profound and cancer-selective role for PRC2 in repressing multiple cytokine pathways. We find that PRC2 represses hundreds of IFNγ stimulated genes (ISGs), cytokines and cytokine receptors. This target repertoire is significantly broadened in cancer vs non-cancer cells, and is distinct in different cancer types. PRC2 is therefore a higher order regulator of the immune program in cancer cells. Inhibiting PRC2 with either RNAi or EZH2 inhibitors activates cytokine/cytokine receptor promoters marked with bivalent H3K27me3/H3K4me3 chromatin, and augments responsiveness to diverse immune signals. PRC2 inhibition rescues immune gene induction even in the absence of SWI/SNF, a tumor suppressor defective in ~20% of human cancers. This novel PRC2 function in tumor cells could profoundly impact the mechanism of action and efficacy of EZH2 inhibitors in cancer treatment.

Utp8p is an essential nucleolar component of the nuclear tRNA export machinery in Saccharomyces cerevisiae. It is thought to act at a step between tRNA maturation/aminoacylation and translocation of the tRNA across the nuclear pore complex. To understand the function of Utp8p in nuclear tRNA export, a comprehensive affinity purification analysis was conducted to identify proteins that interact with Utp8p in vivo. In addition to finding proteins that have been shown previously to copurify with Utp8p, a number of new interactions were identified. These interactions include aminoacyl-tRNA synthetases, the RanGTPase Gsp1p, and nuclear tRNA export receptors such as Los1p and Msn5p. Characterization of the interaction of Utp8p with a subset of the newly identified proteins suggests that Utp8p most likely transfer tRNAs to the nuclear tRNA export receptors by using a channeling mechanism.