In Saccharomyces cerevisiae, deletion of large ribosomal subunit protein-encoding genes increases the replicative lifespan in a Gcn4-dependent manner. However, how Gcn4, a key transcriptional activator of amino acid biosynthesis genes, increases lifespan, is unknown. Here we show that Gcn4 acts as a repressor of protein synthesis. By analyzing the messenger RNA and protein abundance, ribosome occupancy and protein synthesis rate in various yeast strains, we demonstrate that Gcn4 is sufficient to reduce protein synthesis and increase yeast lifespan. Chromatin immunoprecipitation reveals Gcn4 binding not only at genes that are activated, but also at genes, some encoding ribosomal proteins, that are repressed upon Gcn4 overexpression. The promoters of repressed genes contain Rap1 binding motifs. Our data suggest that Gcn4 is a central regulator of protein synthesis under multiple perturbations, including ribosomal protein gene deletions, calorie restriction, and rapamycin treatment, and provide an explanation for its role in longevity and stress response.

RNA interference screen previously revealed that a HECTdomain E3 ubiquitin ligase, neuronal precursor cell expressed, developmentally down-regulated 4-2 (Nedd4-2), is necessary for ubiquitination and endocytosis of the dopamine transporter (DAT) induced by the activation of protein kinase C (PKC). To further confirm the role of Nedd4-2 in DAT ubiquitination and endocytosis, we demonstrated that the depletion of Nedd4-2 by two different small interfering RNA (siRNA) duplexes suppressed PKC-dependent ubiquitination and endocytosis of DAT in human and porcine cells, whereas knock-down of a highly homologous E3 ligase, Nedd4-1, had no effect on DAT. The abolished DAT ubiquitination in Nedd4-2-depleted cells was rescued by expression of recombinant Nedd4-2. Moreover, overexpression of Nedd4-2 resulted in increased PKC-dependent ubiquitination of DAT. Mutational inactivation of the HECT domain of Nedd4-2 inhibited DAT ubiquitination and endocytosis. Structure-function analysis of Nedd4-2-mediated DAT ubiquitination revealed that the intact WW4 domain and to a lesser extent WW3 domain are necessary for PKC-dependent DAT ubiquitination. Moreover, a fragment of the Nedd4-2 molecule containing WW3, WW4, and HECT domains was sufficient for fully potentiating PKC-dependent ubiquitination of DAT. Analysis of DAT ubiquitination using polyubiquitin chain-specific antibodies showed that DAT is mainly conjugated with Lys 63 -linked ubiquitin chains. siRNA analysis demonstrated that this polyubiquitination is mediated by Nedd4-2 cooperation with UBE2D and UBE2L3 E2 ubiquitin-conjugating enzymes. The model is proposed whereby each ubiquitinated DAT molecule is modified by a single four-ubiquitin Lys 63 -linked chain that can be conjugated to various lysine residues of DAT.Post-translational modification of proteins by ubiquitination has recently emerged as an important mechanism governing membrane trafficking processes. Ubiquitin moieties attached to integral membrane proteins serve to target these proteins for rapid internalization at the cell surface and, after internalization, to lysosomes for degradation (1-3). In addition, ubiquitination of the components of internalization and endosomal sorting machineries regulate interactions and turnover of these proteins (1, 4 -5).Ubiquitination is accomplished through sequential actions of the E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzyme, and E3 ubiquitin ligase, the latter typically determining the substrate specificity of the reaction (6 -7). Proteins can be mono-and polyubiquitinated. Polyubiquitin chains can be formed by conjugation of ubiquitin to any of seven lysines in the ubiquitin molecule, although chains linked through Lys We have recently demonstrated that ubiquitination mediates protein kinase C (PKC) 2 -dependent endocytosis of the human dopamine transporter (DAT) (13-15). Upon PKC activation by phorbol esters, heterologously expressed DAT is internalized via a clathrin-dependent endocytic pathway and degraded in lysosomes (16,17). Ubiquitination of thre...

TREX2 is a proofreading 3 ¶-5 ¶ exonuclease that can be involved in genome maintenance; however, its biological role remains undefined. To better understand the function and physiologic relevance of TREX2, we generated mice deficient in TREX2 by targeted disruption of its unique coding exon. The knockout mice are viable and do not show relevant differences in growth, survival, lymphocyte development, or spontaneous tumor incidence compared with their wild-type counterparts over a period of up to 2 years. Also, we did not observe chromosomal instability or defects in cell proliferation and cell cycle upon loss of TREX2. We have observed that TREX2 expression is not ubiquitous, being expressed preferentially in tissues with stratified squamous epithelia, such as the skin or esophagus, and specifically in keratinocytes. Interestingly, TREX2-null mice are more susceptible to skin carcinogenesis induced by 7,12-dimethylbenz(a)anthracene (DMBA) compared with wild-type mice. This phenotype correlates with a reduction of DMBA-induced apoptosis in both the epidermis and keratinocytes of TREX2-null mice. Altogether, our results suggest a tumor suppressor role for TREX2 in skin carcinogenesis through which it contributes to keratinocyte apoptosis under conditions of genotoxic stress. [Cancer Res 2009;69(16):6676-84]

Cationic amino acid transporter 1 (CAT-1) is responsible for the bulk of the uptake of cationic amino acids in most mammalian cells. Activation of protein kinase C (PKC) leads to downregulation of the cell surface CAT-1. To examine the mechanisms of PKC-induced down-regulation of CAT-1, a functional mutant of CAT-1 (CAT-1-HA-GFP) was generated in which a hemagglutinin antigen (HA) epitope tag was introduced into the second extracellular loop and GFP was attached to the carboxyl terminus. CAT-1-HA-GFP was stably expressed in porcine aorthic endothelial and human epithelial kidney (HEK) 293 cells. Using the HA antibody internalization assay we have demonstrated that PKC-dependent endocytosis was strongly inhibited by siRNA depletion of clathrin heavy chain, indicating that CAT-1-HA-GFP internalization requires clathrin-coated pits. Internalized CAT-1-HA-GFP was accumulated in early, recycling, and late endosomes. PKC activation also resulted in ubiquitination of CAT-1. CAT-1 ubiquitination and endocytosis in phorbol ester-stimulated porcine aorthic endothelial and HEK293 cells were inhibited by siRNA knockdown of NEDD4-2 and NEDD4-1 E3 ubiquitin ligases, respectively. In contrast, ubiquitination and endocytosis of the dopamine transporter was dependent on NEDD4-2 in all cell types tested. Altogether, our data suggest that ubiquitination mediated by NEDD4-2 or NEDD4-1 leading to clathrin-mediated endocytosis is the common mode of regulation of various transporter proteins by PKC. Cationic amino acid transporter (CAT)-12 is a member of the SLC7 gene family of membrane transport proteins. The SLC7 family is divided into two groups, glycosylated (CAT) and nonglycosylated transporters (reviewed in Ref. 1). The latter associate with a glycosylated protein from the SLC3 family to form heteromeric amino acid transporters. There are four proven members in the CAT subfamily: CAT-1, CAT-2A, CAT-2B, and CAT-3 (reviewed in Ref. 2). All CATs have a predicted topology of 14 transmembrane domains with cytoplasmic amino and carboxyl termini. The largest, third extracellular loop is glycosylated in all CATs. CAT-2 proteins are also glycosylated in the second loop. CAT-1 is expressed ubiquitously in all adult tissues except the liver (reviewed in Ref.3). The transport properties of CAT-1 resemble those of system y ϩ , defined by the selectivity for cationic amino acids, K m of 0.1-0.2 mM, Na ϩ and pH independence, and strong trans-stimulation. CAT-1 is ubiquitously expressed and the main portal of entry for cationic amino acids into mammalian cells. Homozygous knockout of CAT-1 in mice is postnatally lethal (4). CAT-1 activity has been reported to be regulated through activation of protein kinase C (PKC) (5, 6). Even though there are some discrepancies, most studies conclude that activation of PKC leads to inhibition of L-Arg uptake due to the decreased activity of CAT-1. Studies of CAT-1 phosphorylation and mutations of the PKC phosphorylation consensus sites in CAT-1 suggested that CAT-1 inhibition by PKC is not due to CAT-1 phosp...

High-throughput sequencing has greatly facilitated the discovery of long and short non-coding RNAs (ncRNAs), which frequently guide ribonucleoprotein complexes to RNA targets, to modulate their metabolism and expression. However, for many ncRNAs, the targets remain to be discovered. In this study, we developed computational methods to map C/D box snoRNA target sites using data from core small nucleolar ribonucleoprotein crosslinking and immunoprecipitation and from transcriptome-wide mapping of 2΄-O-ribose methylation sites. We thereby assigned the snoRNA guide to a known methylation site in the 18S rRNA, we uncovered a novel partially methylated site in the 28S ribosomal RNA, and we captured a site in the 28S rRNA in interaction with multiple snoRNAs. Although we also captured mRNAs in interaction with snoRNAs, we did not detect 2΄-O-methylation of these targets. Our study provides an integrated approach to the comprehensive characterization of 2΄-O-methylation targets of snoRNAs in species beyond those in which these interactions have been traditionally studied and contributes to the rapidly developing field of ‘epitranscriptomics’.

AbstractmiRNAs are small RNAs that regulate gene expression post‐transcriptionally. By repressing the translation and promoting the degradation of target mRNAs, miRNAs may reduce the cell‐to‐cell variability in protein expression, induce correlations between target expression levels, and provide a layer through which targets can influence each other's expression as “competing RNAs” (ceRNAs). However, experimental evidence for these behaviors is limited. Combining mathematical modeling with RNA sequencing of individual human embryonic kidney cells in which the expression of two distinct miRNAs was induced over a wide range, we have inferred parameters describing the response of hundreds of miRNA targets to miRNA induction. Individual targets have widely different response dynamics, and only a small proportion of predicted targets exhibit high sensitivity to miRNA induction. Our data reveal for the first time the response parameters of the entire network of endogenous miRNA targets to miRNA induction, demonstrating that miRNAs correlate target expression and at the same time increase the variability in expression of individual targets across cells. The approach is generalizable to other miRNAs and post‐transcriptional regulators to improve the understanding of gene expression dynamics in individual cell types.

Trex2 is a keratinocyte-specific 3'-deoxyribonuclease that participates in the maintenance of skin homeostasis after DNA damage. Here, we show that this exonuclease is strongly upregulated in human psoriasis, a hyperproliferative and inflammatory skin disease. Similarly, the imiquimod (IMQ)- and Il23-induced mouse psoriasis was associated with a substantial upregulation of Trex2, which was recruited into fragmented chromatin in keratinocytes that were undergoing impaired proliferation, differentiation, and cell death, indicating an important role in DNA processing. Using Trex2 knockout mice, we have found that Trex2 deficiency attenuated IMQ-induced psoriasis-like skin inflammation and enhanced IMQ-induced parakeratosis. Also, Il23-induced ear swelling was diminished in Trex2 knockout mice in comparison with wild-type (wt) mice. Transcriptome analysis identified multiple genes that were deregulated by Trex2 loss after treatment with IMQ. Specifically, immune response genes and pathways normally associated with inflammation were downregulated, whereas those related to skin differentiation and chromatin biology showed increased expression. Interestingly, Trex2 deficiency led to decreased IMQ-induced keratinocyte death via both cell autonomous and noncell autonomous mechanisms. Hence, our data indicate that Trex2 acts as a critical factor in the pathogenesis of psoriasis by promoting keratinocyte apoptosis and enucleation and thereby influencing skin immune responses.

The endocytic compartment is emerging as a functional platform for controlling important cellular processes. We have found that ∼10 to 15% of total KRas, a protein that is frequently mutated in cancer, is present on endosomes, independent of its activation state. The dynamics of GFP-KRas wild-type (WT) and constitutively active or inactive mutants on endosomes were analyzed by fluorescence recovery after photobleaching (FRAP) microscopy. The measurements revealed an extraordinarily fast recovery of KRas WT [half-time (HT), ∼1.3 s] compared to HRas, Rab5, and EGFR, with the active KRasG12V mutant being significantly faster and more mobile (HT, ∼1 s, and ∼82% of exchangeable fraction) than the inactive KRasS17N (HT, ∼1.6 s, and ∼60% of exchangeable fraction). KRas rapidly switches from the cytoplasm to the endosomal membranes by an electrostatic interaction between its polybasic region and the endosomal acidic phospholipids, mainly phosphatidylserine.-Gelabert-Baldrich, M., Soriano-Castell, D., Calvo, M., Lu, A., Viña-Vilaseca, A., Rentero, C., Pol, A., Grinstein, S. Enrich, C., Tebar, F. Dynamics of KRas on endosomes: involvement of acidic phospholipids in its association.