Search

Differentiating neutrophils undergo large-scale changes in nuclear morphology. How such alterations in structure are established and modulated upon exposure to microbial agents is largely unknown. Here, we found that prior to encounter with bacteria, an armamentarium of inflammatory genes was positioned in a transcriptionally passive environment suppressing premature transcriptional activation. Upon microbial exposure, however, human neutrophils rapidly (

Alternative lengthening of telomeres (ALT) is a telomerase-independent telomere maintenance mechanism that occurs in a subset of cancers. One of the hallmarks of ALT cancer is the excessively clustered telomeres in promyelocytic leukemia (PML) bodies, represented as large bright telomere foci. Here, we present a model system that generates telomere clustering in nuclear polySUMO (small ubiquitin-like modification)/polySIM (SUMO-interacting motif) condensates, analogous to PML bodies, and thus artificially engineered ALT-associated PML body (APB)-like condensates in vivo. We observed that the ALT-like phenotypes (i.e., a small fraction of heterogeneous telomere lengths and formation of C circles) are rapidly induced by introducing the APB-like condensates together with BLM through its helicase domain, accompanied by ssDNA generation and RPA accumulation at telomeres. Moreover, these events lead to mitotic DNA synthesis (MiDAS) at telomeres mediated by RAD52 through its highly conserved N-terminal domain. We propose that the clustering of large amounts of telomeres in human cancers promotes ALT that is mediated by MiDAS, analogous to Saccharomyces cerevisiae type II ALT survivors.

The exosome functions in the degradation of diverse RNA species, yet how it is negatively regulated remains largely unknown. Here, we show that NRDE2 forms a 1:1 complex with MTR4, a nuclear exosome cofactor critical for exosome recruitment, via a conserved MTR4-interacting domain (MID). Unexpectedly, NRDE2 mainly localizes in nuclear speckles, where it inhibits MTR4 recruitment and RNA degradation, and thereby ensures efficient mRNA nuclear export. Structural and biochemical data revealed that NRDE2 interacts with MTR4's key residues, locks MTR4 in a closed conformation, and inhibits MTR4 interaction with the exosome as well as proteins important for MTR4 recruitment, such as the cap-binding complex (CBC) and ZFC3H1. Functionally, MID deletion results in the loss of self-renewal of mouse embryonic stem cells. Together, our data pinpoint NRDE2 as a nuclear exosome negative regulator that ensures mRNA stability and nuclear export.

The total number of nuclear pore complexes (NPCs) per nucleus varies greatly between different cell types and is known to change during cell differentiation and cell transformation. However, the underlying mechanisms that control how many nuclear transport channels are assembled into a given nuclear envelope remain unclear. Here, we report that depletion of the NPC basket protein Tpr, but not Nup153, dramatically increases the total NPC number in various cell types. This negative regulation of Tpr occurs via a phosphorylation cascade of extracellular signal-regulated kinase (ERK), the central kinase of the mitogen-activated protein kinase (MAPK) pathway. Tpr serves as a scaffold for ERK to phosphorylate the nucleoporin (Nup) Nup153, which is critical for early stages of NPC biogenesis. Our results reveal a critical role of the Nup Tpr in coordinating signal transduction pathways during cell proliferation and the dynamic organization of the nucleus.

Alternative premessenger RNA (pre-mRNA) splicing is a post-transcriptional mechanism for controlling gene expression. Splicing patterns are determined by both RNA-binding proteins and nuclear pre-mRNA structure. Here, we analyzed pre-mRNA splicing patterns, RNA-binding sites, and RNA structures near these binding sites coordinately controlled by two splicing factors: the heterogeneous nuclear ribonucleoprotein hnRNPA1 and the RNA helicase DDX5. We identified thousands of alternative pre-mRNA splicing events controlled by these factors by RNA sequencing (RNA-seq) following RNAi. Enhanced cross-linking and immunoprecipitation (eCLIP) on nuclear extracts was used to identify protein–RNA-binding sites for both proteins in the nuclear transcriptome. We found a significant overlap between hnRNPA1 and DDX5 splicing targets and that they share many closely linked binding sites as determined by eCLIP analysis. In vivo SHAPE (selective 2′-hydroxyl acylation analyzed by primer extension) chemical RNA structure probing data were used to model RNA structures near several exons controlled and bound by both proteins. Both sequence motifs and in vivo UV cross-linking sites for hnRNPA1 and DDX5 were used to map binding sites in their RNA targets, and often these sites flanked regions of higher chemical reactivity, suggesting an organized nature of nuclear pre-mRNPs. This work provides a first glimpse into the possible RNA structures surrounding pre-mRNA splicing factor-binding sites.

In eukaryotes, antisense transcription can regulate sense transcription by induction of epigenetic modifications. We showed previously that antisense transcription triggers Dicer-independent siRNA (disiRNA) production and disiRNA locus DNA methylation (DLDM) in Neurospora crassa. Here we show that the conserved exonuclease ERI-1 (enhanced RNAi-1) is a critical component in this process. Antisense transcription and ERI-1 binding to target RNAs are necessary and sufficient to trigger DLDM. Convergent transcription causes stalling of RNA polymerase II during transcription, which permits ERI-1 to bind nascent RNAs in the nucleus and recruit a histone methyltransferase complex that catalyzes chromatin modifications. Furthermore, we show that, in the cytoplasm, ERI-1 targets hundreds of transcripts from loci without antisense transcription to regulate RNA stability. Together, our results demonstrate a critical role for transcription kinetics in long noncoding RNA-mediated epigenetic modifications and identify ERI-1 as an important regulator of cotranscriptional gene silencing and post-transcriptional RNA metabolism.

N-terminal acetylation of the first two amino acids on proteins is a prevalent cotranslational modification. Despite its abundance, the biological processes associated with this modification are not well understood. Here, we mapped the pattern of protein N-terminal acetylation in Caenorhabditis elegans, uncovering a conserved set of rules for this protein modification and identifying substrates for the N-terminal acetyltransferase B (NatB) complex. We observed an enrichment for global protein N-terminal acetylation and also specifically for NatB substrates in the nucleus, supporting the importance of this modification for regulating biological functions within this cellular compartment. Peptide profiling analysis provides evidence of cross-talk between N-terminal acetylation and internal modifications in a NAT substrate-specific manner. In vivo studies indicate that N-terminal acetylation is critical for meiosis, as it regulates the assembly of the synaptonemal complex (SC), a proteinaceous structure ubiquitously present during meiosis from yeast to humans. Specifically, N-terminal acetylation of NatB substrate SYP-1, an SC structural component, is critical for SC assembly. These findings provide novel insights into the biological functions of N-terminal acetylation and its essential role during meiosis.

Some mitochondrial long noncoding RNAs (lncRNAs) are encoded by nuclear DNA, but the mechanisms that mediate their transport to mitochondria are poorly characterized. Using affinity RNA pull-down followed by mass spectrometry analysis, we found two RNA-binding proteins (RBPs), HuR (human antigen R) and GRSF1 (G-rich RNA sequence-binding factor 1), that associated with the nuclear DNA-encoded lncRNA RMRP and mobilized it to mitochondria. In cultured human cells, HuR bound RMRP in the nucleus and mediated its CRM1 (chromosome region maintenance 1)-dependent export to the cytosol. After RMRP was imported into mitochondria, GRSF1 bound RMRP and increased its abundance in the matrix. Loss of GRSF1 lowered the mitochondrial levels of RMRP, in turn suppressing oxygen consumption rates and modestly reducing mitochondrial DNA replication priming. Our findings indicate that RBPs HuR and GRSF1 govern the cytoplasmic and mitochondrial localization of the lncRNA RMRP, which is encoded by nuclear DNA but has key functions in mitochondria.

Nucleoporins (Nups) are a family of proteins best known as the constituent building blocks of nuclear pore complexes (NPCs), membrane-embedded channels that mediate nuclear transport across the nuclear envelope. Recent evidence suggests that several Nups have additional roles in controlling the activation and silencing of developmental genes; however, the mechanistic details of these functions remain poorly understood. Here, we show that depletion of Nup153 in mouse embryonic stem cells (mESCs) causes the derepression of developmental genes and induction of early differentiation. This loss of stem cell identity is not associated with defects in the nuclear import of key pluripotency factors. Rather, Nup153 binds around the transcriptional start site (TSS) of developmental genes and mediates the recruitment of the polycomb-repressive complex 1 (PRC1) to a subset of its target loci. Our results demonstrate a chromatin-associated role of Nup153 in maintaining stem cell pluripotency by functioning in mammalian epigenetic gene silencing.

Bidirectional tRNA movement between the nucleus and the cytoplasm serves multiple biological functions. To gain a biochemical understanding of the mechanisms for tRNA subcellular dynamics, we developed in vivo β-importin complex coimmunoprecipitation (co-IP) assays using budding yeast. Our studies provide the first in vivo biochemical evidence that two β-importin family members, Los1 (exportin-t) and Msn5 (exportin-5), serve overlapping but distinct roles in tRNA nuclear export. Los1 assembles complexes with RanGTP and tRNA. Both intron-containing pre-tRNAs and spliced tRNAs, regardless of whether they are aminoacylated, assemble into Los1–RanGTP complexes, documenting that Los1 participates in both primary nuclear export and re-export of tRNAs to the cytoplasm. In contrast, β-importin Msn5 preferentially assembles with RanGTP and spliced, aminoacylated tRNAs, documenting its role in tRNA nuclear re-export. Tef1/2 (the yeast form of translation elongation factor 1α [eEF1A]) aids the specificity of Msn5 for aminoacylated tRNAs to form a quaternary complex consisting of Msn5, RanGTP, aminoacylated tRNA, and Tef1/2. Assembly and/or stability of this quaternary complex requires Tef1/2, thereby facilitating efficient re-export of aminoacylated tRNAs to the cytoplasm.

In many cells, mRNAs containing inverted repeated Alu elements (IRAlus) in their 3' untranslated regions (UTRs) are inefficiently exported to the cytoplasm. Such nuclear retention correlates with paraspeckle-associated protein complexes containing p54(nrb). However, nuclear retention of mRNAs containing IRAlus is variable, and how regulation of retention and export is achieved is poorly understood. Here we show one mechanism of such regulation via the arginine methyltransferase CARM1 (coactivator-associated arginine methyltransferase 1). We demonstrate that disruption of CARM1 enhances the nuclear retention of mRNAs containing IRAlus. CARM1 regulates this nuclear retention pathway at two levels: CARM1 methylates the coiled-coil domain of p54(nrb), resulting in reduced binding of p54(nrb) to mRNAs containing IRAlus, and also acts as a transcription regulator to suppress NEAT1 transcription, leading to reduced paraspeckle formation. These actions of CARM1 work together synergistically to regulate the export of transcripts containing IRAlus from paraspeckles under certain cellular stresses, such as poly(I:C) treatment. This work demonstrates how a post-translational modification of an RNA-binding protein affects protein-RNA interaction and also uncovers a mechanism of transcriptional regulation of the long noncoding RNA NEAT1.

Mutations in CTC1 lead to the telomere syndromes Coats Plus and dyskeratosis congenita (DC), but the molecular mechanisms involved remain unknown. CTC1 forms with STN1 and TEN1 a trimeric complex termed CST, which binds ssDNA, promotes telomere DNA synthesis, and inhibits telomerase-mediated telomere elongation. Here we identify CTC1 disease mutations that disrupt CST complex formation, the physical interaction with DNA polymerase α-primase (polα-primase), telomeric ssDNA binding in vitro, accumulation in the nucleus, and/or telomere association in vivo. While having diverse molecular defects, CTC1 mutations commonly lead to the accumulation of internal single-stranded gaps of telomeric DNA, suggesting telomere DNA replication defects as a primary cause of the disease. Strikingly, mutations in CTC1 may also unleash telomerase repression and telomere length control. Hence, the telomere defect initiated by CTC1 mutations is distinct from the telomerase insufficiencies seen in classical forms of telomere syndromes, which cause short telomeres due to reduced maintenance of distal telomeric ends by telomerase. Our analysis provides molecular evidence that CST collaborates with DNA polα-primase to promote faithful telomere DNA replication.

Senescence is a stress-responsive form of stable cell cycle exit. Senescent cells have a distinct gene expression profile, which is often accompanied by the spatial redistribution of heterochromatin into senescence-associated heterochromatic foci (SAHFs). Studying a key component of the nuclear lamina lamin B1 (LMNB1), we report dynamic alterations in its genomic profile and their implications for SAHF formation and gene regulation during senescence. Genome-wide mapping reveals that LMNB1 is depleted during senescence, preferentially from the central regions of lamina-associated domains (LADs), which are enriched for Lys9 trimethylation on histone H3 (H3K9me3). LMNB1 knockdown facilitates the spatial relocalization of perinuclear H3K9me3-positive heterochromatin, thus promoting SAHF formation, which could be inhibited by ectopic LMNB1 expression. Furthermore, despite the global reduction in LMNB1 protein levels, LMNB1 binding increases during senescence in a small subset of gene-rich regions where H3K27me3 also increases and gene expression becomes repressed. These results suggest that LMNB1 may contribute to senescence in at least two ways due to its uneven genome-wide redistribution: first, through the spatial reorganization of chromatin and, second, through gene repression.

Sex is determined in Caenorhabditis elegans by the ratio of X chromosomes to the sets of autosomes, the X:A signal. A set of genes called X signal elements (XSEs) communicates X-chromosome dose by repressing the masculinizing sex determination switch gene xol-1 (XO lethal) in a dose-dependent manner. xol-1 is active in 1X:2A embryos (males) but repressed in 2X:2A embryos (hermaphrodites). Here we showed that the autosome dose is communicated by a set of autosomal signal elements (ASEs) that act in a cumulative, dose-dependent manner to counter XSEs by stimulating xol-1 transcription. We identified new ASEs and explored the biochemical basis by which ASEs antagonize XSEs to determine sex. Multiple antagonistic molecular interactions carried out on a single promoter explain how different X:A values elicit different sexual fates. XSEs (nuclear receptors and homeodomain proteins) and ASEs (T-box and zinc finger proteins) bind directly to several sites on xol-1 to counteract each other's activities and thereby regulate xol-1 transcription. Disrupting ASE- and XSE-binding sites in vivo recapitulated the misregulation of xol-1 transcription caused by disrupting cognate signal element genes. XSE- and ASE-binding sites are distinct and nonoverlapping, suggesting that direct competition for xol-1 binding is not how XSEs counter ASEs. Instead, XSEs likely antagonize ASEs by recruiting cofactors with reciprocal activities that induce opposite transcriptional states. Most ASE- and XSE-binding sites overlap xol-1's −1 nucleosome, which carries activating chromatin marks only when xol-1 is turned on. Coactivators and corepressors tethered by proteins similar to ASEs and XSEs are known to deposit and remove such marks. The concept of a sex signal comprising competing XSEs and ASEs arose as a theory for fruit flies a century ago. Ironically, while the recent work of others showed that the fly sex signal does not fit this simple paradigm, our work shows that the worm signal does.

Endoplasmic reticulum (ER) stress is emerging as a potential contributor to the onset of type 2 diabetes by making cells insulin-resistant. However, our understanding of the mechanisms by which ER stress affects insulin response remains fragmentary. Here we present evidence that the ER stress pathway acts via a conserved signaling mechanism involving the protein kinase PERK to modulate cellular insulin responsiveness. Insulin signaling via AKT reduces activity of FOXO transcription factors. In some cells, PERK can promote insulin responsiveness. However, we found that PERK also acts oppositely via phosphorylation of FOXO to promote FOXO activity. Inhibition of PERK improves cellular insulin responsiveness at the level of FOXO activity. We suggest that the protein kinase PERK may be a promising pharmacological target for ameliorating insulin resistance.

Nuclear migration during yeast karyogamy, termed nuclear congression, is required to initiate nuclear fusion. Congression involves a specific regulation of the microtubule minus end-directed kinesin-14 motor Kar3 and a rearrangement of the cytoplasmic microtubule attachment sites at the spindle pole bodies (SPBs). However, how these elements interact to produce the forces necessary for nuclear migration is less clear. We used electron tomography, molecular genetics, quantitative imaging, and first principles modeling to investigate how cytoplasmic microtubules are organized during nuclear congression. We found that Kar3, with the help of its light chain, Cik1, is anchored during mating to the SPB component Spc72 that also serves as a nucleator and anchor for microtubules via their minus ends. Moreover, we show that no direct microtubule–microtubule interactions are required for nuclear migration. Instead, SPB-anchored Kar3 exerts the necessary pulling forces laterally on microtubules emanating from the SPB of the mating partner nucleus. Therefore, a twofold symmetrical application of the core principle that drives nuclear migration in higher cells is used in yeast to drive nuclei toward each other before nuclear fusion.

To compare nuclear and cytoplasmic RNA from a single cell type, free of cross-contamination, we studied the oocyte of the frog Xenopus tropicalis, a giant cell with an equally giant nucleus. We isolated RNA from manually dissected nuclei and cytoplasm of mature oocytes and subjected it to deep sequencing. Cytoplasmic mRNA consisted primarily of spliced exons derived from ∼6700 annotated genes. Nearly all of these genes were represented in the nucleus by intronic sequences. However, unspliced nascent transcripts were not detected. Inhibition of transcription or splicing for 1–2 d had little or no effect on the abundance of nuclear intronic sequences, demonstrating that they are unusually stable. RT–PCR analysis showed that these stable intronic sequences are transcribed from the coding strand and that a given intron can be processed into more than one molecule. Stable intronic sequence RNA (sisRNA) from the oocyte nucleus constitutes a new class of noncoding RNA. sisRNA is detectable by RT–PCR in samples of total RNA from embryos up to the mid-blastula stage, when zygotic transcription begins. Storage of sisRNA in the oocyte nucleus and its transmission to the developing embryo suggest that it may play important regulatory roles during oogenesis and/or early embryogenesis.

The molecular mechanisms for target mRNA degradation in Caenorhabditis elegans undergoing RNAi are not fully understood. Using a combination of genetic, proteomic, and biochemical approaches, we report a divergent RDE-10/RDE-11 complex that is required for RNAi in C. elegans. Genetic analysis indicates that the RDE-10/RDE-11 complex acts in parallel to nuclear RNAi. Association of the complex with target mRNA is dependent on RDE-1 but not RRF-1, suggesting that target mRNA recognition depends on primary but not secondary siRNA. Furthermore, RDE-11 is required for mRNA degradation subsequent to target engagement. Deep sequencing reveals a fivefold decrease in secondary siRNA abundance in rde-10 and rde-11 mutant animals, while primary siRNA and microRNA biogenesis is normal. Therefore, the RDE-10/RDE-11 complex is critical for amplifying the exogenous RNAi response. Our work uncovers an essential output of the RNAi pathway in C. elegans.

Macrophages play critical roles in events ranging from host defense to obesity and cancer, where they infiltrate affected tissues and orchestrate immune responses in tandem with the remodeling of the extracellular matrix (ECM). Despite the dual roles played by macrophages in inflammation, the functions of macrophage-derived proteinases are typically relegated to tissue-invasive or -degradative events. Here we report that the membrane-tethered matrix metalloenzyme MT1-MMP not only serves as an ECM-directed proteinase, but unexpectedly controls inflammatory gene responses wherein MT1-MMP−/− macrophages mount exaggerated chemokine and cytokine responses to immune stimuli both in vitro and in vivo. MT1-MMP modulates inflammatory responses in a protease-independent fashion in tandem with its trafficking to the nuclear compartment, where it triggers the expression and activation of a phosphoinositide 3-kinase δ (PI3Kδ)/Akt/GSK3β signaling cascade. In turn, MT1-MMP-dependent PI3Kδ activation regulates the immunoregulatory Mi-2/NuRD nucleosome remodeling complex that is responsible for controlling macrophage immune response. These findings identify a novel role for nuclear MT1-MMP as a previously unsuspected transactivator of signaling networks central to macrophage immune responses.

Hundreds of microRNAs (miRNAs) have been discovered in metazoans and plants, and understanding of their biogenesis has advanced dramatically; however, relatively little is known about the cofactors necessary for miRNA regulation of target gene expression. In Caenorhabditis elegans, the conserved miRNA let-7 and its paralogs, including mir-84, control the timing of stage-specific developmental events. To identify factors required for the activity of mir-84 and possibly other miRNAs, we screened for mutations that suppress the developmental defects caused by overexpression of mir-84. Mutations in the somi-1 gene prevent these defects without affecting the expression level of mir-84. Loss of somi-1 also causes phenotypes similar to deletion of mir-84, showing that somi-1 is necessary for the normal function of this miRNA. somi-1 encodes a zinc finger protein that localizes to nuclear foci and binds the promoters of let-60/RAS, lin-14, and lin-28, genes that may be targeted by mir-84 and similar miRNAs. Genetic evidence shows that somi-1 inhibits lin-14 and induction of the vulval precursors by the let-60/RAS pathway. Proteomic and genetic screens identified conserved chromatin-remodeling and homeodomain transcription factor complexes that work with somi-1 to regulate differentiation. Our results suggest that somi-1 coordinates a nuclear response that complements the activity of mir-84.

Essential messenger RNA (mRNA) export factors execute critical steps to mediate directional transport through nuclear pore complexes (NPCs). At cytoplasmic NPC filaments, the ATPase activity of DEAD-box protein Dbp5 is activated by inositol hexakisphosphate (IP6)-bound Gle1 to mediate remodeling of mRNA–protein (mRNP) complexes. Whether a single Dbp5 executes multiple remodeling events and how Dbp5 is recycled are unknown. Evidence suggests that Dbp5 binding to Nup159 is required for controlling interactions with Gle1 and the mRNP. Using in vitro reconstitution assays, we found here that Nup159 is specifically required for ADP release from Dbp5. Moreover, Gle1-IP6 stimulates ATP binding, thus priming Dbp5 for RNA loading. In vivo, a dbp5-R256D/R259D mutant with reduced ADP binding bypasses the need for Nup159 interaction. However, NPC spatial control is important, as a dbp5-R256D/R259D nup42Δ double mutant is temperature-sensitive for mRNA export. Further analysis reveals that remodeling requires a conformational shift to the Dbp5–ADP form. ADP release factors for DEAD-box proteins have not been reported previously and reflect a new paradigm for regulation. We propose a model wherein Nup159 and Gle1-IP6 regulate Dbp5 cycles by controlling its nucleotide-bound state, allowing multiple cycles of mRNP remodeling by a single Dbp5 at the NPC.

Δ40p53 is a transactivation-deficient isoform of the tumor suppressor p53. We discovered that Δ40p53, in addition to being highly expressed in embryonic stem cells (ESCs), is the major p53 isoform during early stages of embryogenesis in the mouse. By altering the dose of Δ40p53 in ESCs, we identified a critical role for this isoform in maintaining the ESC state. Haploinsufficiency for Δ40p53 causes a loss of pluripotency in ESCs and acquisition of a somatic cell cycle, while increased dosage of Δ40p53 prolongs pluripotency and inhibits progression to a more differentiated state. Δ40p53 controls the switch from pluripotent ESCs to differentiated somatic cells by controlling the activity of full-length p53 at critical targets such as Nanog and the IGF-1 receptor (IGF-1R). The IGF axis plays a central role in the switch between pluripotency and differentiation in ESCs—and Δ40p53, by controlling the level of the IGF-1R, acts as a master regulator of this switch. We propose that this is the primary function of Δ40p53 in cells of the early embryo and stem cells, which are the only normal cells in which this isoform is expressed.

The transcriptional program orchestrated by Hedgehog signaling depends on the Gli family of transcription factors. Gli proteins can be converted to either transcriptional activators or truncated transcriptional repressors. We show that the interaction between Gli3 and Suppressor of Fused (Sufu) regulates the formation of either repressor or activator forms of Gli3. In the absence of signaling, Sufu restrains Gli3 in the cytoplasm, promoting its processing into a repressor. Initiation of signaling triggers the dissociation of Sufu from Gli3. This event prevents formation of the repressor and instead allows Gli3 to enter the nucleus, where it is converted into a labile, differentially phosphorylated transcriptional activator. This key dissociation event depends on Kif3a, a kinesin motor required for the function of primary cilia. We propose that the Sufu–Gli3 interaction is a major control point in the Hedgehog pathway, a pathway that plays important roles in both development and cancer.

During or right after mRNA export via the nuclear pore complex (NPC) in mammalian cells, mRNAs undergo translation mediated by nuclear cap-binding proteins 80 and 20 (CBP80/20). After CBP80/20-dependent translation, CBP80/20 is replaced by cytoplasmic cap-binding protein eIF4E, which directs steady-state translation. Nonsense-mediated mRNA decay (NMD), one of the best-characterized mRNA surveillance mechanisms, has been shown to occur on CBP80/20-bound mRNAs. However, despite the tight link between CBP80/20-dependent translation and NMD, the underlying molecular mechanism and cellular factors that mediate CBP80/20-dependent translation remain obscure. Here, we identify a new MIF4G domain-containing protein, CTIF (CBP80/20-dependent translation initiation factor). CTIF interacts directly with CBP80 and is part of the CBP80/20-dependent translation initiation complex. Depletion of endogenous CTIF from an in vitro translation system selectively blocks the translation of CBP80-bound mRNAs, while addition of purified CTIF restores it. Accordingly, down-regulation of endogenous CTIF abrogates NMD. Confocal microscopy shows that CTIF is localized to the perinuclear region. Our observations demonstrate the existence of CBP80/20-dependent translation and support the idea that CBP80/20-dependent translation is mechanistically different from steady-state translation through identification of a specific cellular protein, CTIF.

The perinuclear localization of Saccharomyces cerevisiae telomeres provides a useful model for studying mechanisms that control chromosome positioning. Telomeres tend to be localized at the nuclear periphery during early interphase, but following S phase they delocalize and remain randomly positioned within the nucleus. We investigated whether DNA replication causes telomere delocalization from the nuclear periphery. Using live-cell fluorescence microscopy, we show that delaying DNA replication causes a corresponding delay in the dislodgment of telomeres from the nuclear envelope, demonstrating that replication of individual telomeres causes their delocalization. Telomere delocalization is not simply the result of recruitment to a replication factory in the nuclear interior, since we found that telomeric DNA replication can occur either at the nuclear periphery or in the nuclear interior. The telomere-binding complex Ku is one of the factors that localizes telomeres to the nuclear envelope. Using a gene locus tethering assay, we show that Ku-mediated peripheral positioning is switched off after DNA replication. Based on these findings, we propose that DNA replication causes telomere delocalization by triggering stable repression of the Ku-mediated anchoring pathway. In addition to maintaining genetic information, DNA replication may therefore regulate subnuclear organization of chromatin.

In Neurospora crassa, a transcription factor, WCC, activates the transcription of frq. FRQ forms homodimers as well as complexes with an RNA helicase, FRH, and the WCC, and translocates into the nucleus to inactivate the WCC, closing the time-delayed negative feedback loop. The detailed mechanism for closing this loop, however, remains incompletely understood. In particular within the nucleus, the low amount of FRQ compared with that of WC-1 creates a conundrum: How can the nuclear FRQ inactivate the larger amount of WCC? One possibility is that FRQ might function as a catalytic component in phosphorylation-dependent inhibition. However, in silico experiments reveal that stoichiometric noncatalytic binding and inhibition can generate a robust oscillator, even when nuclear FRQ levels are substantially lower than nuclear WCC, so long as there is FRQ-dependent clearance of WC-1 from the nucleus. Based on this model, we can predict and now demonstrate that WC-1 stability cycles, that WC-1 is stable in the absence of FRQ, and that physical binding between FRQ and WCC is essential for closure of the negative feedback loop. Moreover, and consistent with a noncatalytic clearance-based model for inhibition, appreciable amounts of the nuclear FRQ:WCC complex accumulate at some times of day, comprising as much as 10% of the nuclear WC-1.

Caenorhabditis elegans SIR-2.1, a member of the sirtuin family related to Saccharomyces cerevisiae Sir2p, has previously been implicated in aging. The mammalian homolog SIRT1 plays important roles in multiple cellular processes including transcriptional repression and stress response. We show that sir-2.1 is essential for the execution of apoptosis in response to DNA damage, and that sir-2.1 genetically acts in parallel to the worm p53-like gene cep-1. This novel cep-1-independent proapoptotic pathway does not require the daf-16 FOXO transcription factor. Cytological analysis of SIR-2.1 suggests a novel mechanism of apoptosis induction. During apoptosis SIR-2.1 changes its subcellular localization from the nucleus to the cytoplasm and transiently colocalizes with the C. elegans Apaf-1 homolog CED-4 at the nuclear periphery. SIR-2.1 translocation is an early event in germ cell apoptosis and is independent of apoptosis execution and cep-1, raising the possibility that SIR-2.1 translocation is linked to the induction of DNA damage-induced apoptosis.

A predominantly nuclear RNA-binding protein, HuR translocates to the cytoplasm in response to stress and proliferative signals, where it stabilizes or modulates the translation of target mRNAs. Here, we present evidence that HuR phosphorylation at S202 by the G2-phase kinase Cdk1 influences its subcellular distribution. HuR was specifically phosphorylated in synchronous G2-phase cultures; its cytoplasmic levels increased by Cdk1-inhibitory interventions and declined in response to Cdk1-activating interventions. In keeping with the prominently cytoplasmic location of the nonphosphorylatable point mutant HuR(S202A), phospho-HuR(S202) was shown to be predominantly nuclear using a novel anti-phospho-HuR(S202) antibody. The enhanced cytoplasmic presence of unphosphorylated HuR was linked to its decreased association with 14–3–3 and to its heightened binding to target mRNAs. Our findings suggest that Cdk1 phosphorylates HuR during G2, thereby helping to retain it in the nucleus in association with 14–3–3 and hindering its post-transcriptional function and anti-apoptotic influence.

Recent studies suggest that increased mitochondrial metabolism and the concomitant decrease in NADH levels mediate calorie restriction (CR)-induced life span extension. The mitochondrial inner membrane is impermeable to NAD (nicotinamide adenine dinucleotide, oxidized form) and NADH, and it is unclear how CR relays increased mitochondrial metabolism to multiple cellular pathways that reside in spatially distinct compartments. Here we show that the mitochondrial components of the malate–aspartate NADH shuttle (Mdh1 [malate dehydrogenase] and Aat1 [aspartate amino transferase]) and the glycerol-3-phosphate shuttle (Gut2, glycerol-3-phosphate dehydrogenase) are novel longevity factors in the CR pathway in yeast. Overexpressing Mdh1, Aat1, and Gut2 extend life span and do not synergize with CR. Mdh1 and Aat1 overexpressions require both respiration and the Sir2 family to extend life span. The mdh1Δaat1Δ double mutation blocks CR-mediated life span extension and also prevents the characteristic decrease in the NADH levels in the cytosolic/nuclear pool, suggesting that the malate–aspartate shuttle plays a major role in the activation of the downstream targets of CR such as Sir2. Overexpression of the NADH shuttles may also extend life span by increasing the metabolic fitness of the cells. Together, these data suggest that CR may extend life span and ameliorate age-associated metabolic diseases by activating components of the NADH shuttles.

Deregulation of cyclin D1 occurs in numerous human cancers through mutations, alternative splicing, and gene amplification. Although cancer-derived cyclin D1 mutants are potent oncogenes in vitro and in vivo, the mechanisms whereby they contribute to neoplasia are poorly understood. We now provide evidence derived from both mouse models and human cancer-derived cells revealing that nuclear accumulation of catalytically active mutant cyclin D1/CDK4 complexes triggers DNA rereplication, resulting from Cdt1 stabilization, which in turn triggers the DNA damage checkpoint and p53-dependent apoptosis. Loss of p53 through mutations or targeted deletion results in increased genomic instability and neoplastic growth. Collectively, the data presented reveal mechanistic insights into how uncoupling of critical cell cycle regulatory events will perturb DNA replication fidelity, thereby contributing to neoplastic transformation.

Silencing within the yeast ribosomal DNA (rDNA) repeats protects the integrity of this highly repetitive array by inhibiting hyperrecombination and repressing transcription from foreign promoters. Using affinity purification combined with highly sensitive mixture mass spectrometry, we have analyzed the protein interaction network involved in suppressing homologous recombination within the rDNA locus. We show that the Net1 and Sir2 subunits of the RENT (regulator of nucleolar silencing and telophase exit) silencing complex, and Fob1, which recruits RENT to the nontranscribed spacer I (NTS1) region of rDNA, are physically associated with Tof2. In addition to RENT components and Fob1, Tof2 copurified with a two-subunit complex composed of Lrs4 and Csm1. Tof2, Lrs4, and Csm1 are recruited to the NTS1 region by Fob1 and are specifically required for silencing at this rDNA region. Moreover, Lrs4 and Csm1 act synergistically with Sir2 to suppress unequal crossover at the rDNA and are released from the nucleolus during anaphase. Together with previous observations showing that Csm1 physically associates with cohesin, these findings suggest a possible model in which RENT, Tof2, and Lrs4/Csm1 physically clamp rDNA to the cohesin ring, thereby restricting the movement of rDNA sister chromatids relative to each other to inhibit unequal exchange.

The periodic destruction of mitotic cyclins is triggered by the activation of the anaphase-promoting complex/cyclosome (APC/C) in mitosis. Although the ability of the APC/C to recognize destruction box (D-box) substrates oscillates throughout the cell cycle, the mechanism regulating APC/C binding to D-box substrates remains unclear. Here, we show that the APC/C inhibitor Emi1 tightly binds both the APC/C and its Cdh1 activator, binds to the D-box receptor site on the APC/CCdh1, and competes with APC/C substrates for D-box binding. Emi1 itself contains a conserved C-terminal D-box, which provides APC/C-binding affinity, and a conserved zinc-binding region (ZBR), which antagonizes APC/C E3 ligase activity independent of tight APC binding. Mutation of the ZBR converts Emi1 into a D-box-dependent APC/C substrate. The identification of a direct Emi1–APC/C complex further explains how Emi1 functions as a stabilizing factor for cyclin accumulation and the need to destroy Emi1 for APC/C activation in mitosis. The combination of a degron/E3 recognition site and an anti-ligase function in Emi1 suggests a general model for how E3 substrates evolve to become pseudosubstrate inhibitors.

Frequency (FRQ) is a central component of interconnected negative and positive limbs of feedback loops of the circadian clock of Neurospora. In the negative limb, FRQ inhibits its transcriptional activator White Collar Complex (WCC) and in the positive limb, FRQ supports accumulation of WCC. We show that these conflicting functions are confined to distinct subcellular compartments and coordinated in temporal fashion. Inactivation of the transcriptional activator WCC requires nuclear FRQ and occurs early after the onset of FRQ expression. Support of WCC accumulation requires cytosolic FRQ and occurs on a post-translational level, when high amounts of FRQ have accumulated. The transcriptional function of FRQ in the negative loop and its post-translational function in the positive loop are independent and associated with distinct regions of FRQ. Phosphorylation of FRQ at the PEST-2 region triggers its maturation from a nuclear repressor toward a cytoplasmic activator.

Chromatin changes within the context of DNA repair remain largely obscure. Here we show that DNA damage induces monoubiquitylation of histone H2A in the vicinity of DNA lesions. Ultraviolet (UV)-induced monoubiquitylation of H2A is dependent on functional nucleotide excision repair and occurs after incision of the damaged strand. The ubiquitin ligase Ring2 is required for the DNA damage-induced H2A ubiquitylation. UV-induced ubiquitylation of H2A is dependent on the DNA damage signaling kinase ATR (ATM- and Rad3-related) but not the related kinase ATM (ataxia telangiectasia-mutated). Although the response coincides with phosphorylation of variant histone H2AX, H2AX was not required for H2A ubiquitylation. Together our data show that monoubiquitylation of H2A forms part of the cellular response to UV damage and suggest a role of this modification in DNA repair-induced chromatin remodeling.

The cytokine-activated Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway plays an important role in the control of a wide variety of biological processes. When misregulated, JAK/STAT signaling is associated with various human diseases, such as immune disorders and tumorigenesis. To gain insights into the mechanisms by which JAK/STAT signaling participates in these diverse biological responses, we carried out a genome-wide RNA interference (RNAi) screen in cultured Drosophila cells. We identified 121 genes whose double-stranded RNA (dsRNA)-mediated knockdowns affected STAT92E activity. Of the 29 positive regulators, 13 are required for the tyrosine phosphorylation of STAT92E. Furthermore, we found that the Drosophila homologs of RanBP3 and RanBP10 are negative regulators of JAK/STAT signaling through their control of nucleocytoplasmic transport of STAT92E. In addition, we identified a key negative regulator of Drosophila JAK/STAT signaling, protein tyrosine phosphatase PTP61F, and showed that it is a transcriptional target of JAK/STAT signaling, thus revealing a novel negative feedback loop. Our study has uncovered many uncharacterized genes required for different steps of the JAK/STAT signaling pathway.

Upon activation by far-red light, phytochrome A signals are transduced through several pathways to promote photomorphogenesis. The COP1 E3 ligase represses photomorphogenesis in part by targeting transcription activators such as LAF1 and HY5 for destruction. Another positive regulator of photomorphogenesis is HFR1, a basic helix-loop-helix (bHLH) transcription factor. Here, we show that HFR1 colocalizes with COP1 in nuclear bodies, and that the HFR1 N-terminal region (amino acids 1-131) interacts with the COP1 WD40 domain. HFR1(ΔN), an HFR1 mutant lacking the two N-terminal, COP1-interacting motifs, still localizes in nuclear bodies and retains weak affinity for COP1. Both HFR1 and HFR1(ΔN) can be ubiquitinated by COP1, although with different efficiencies. Expression of 35S-HFR1(ΔN) in wild-type plants confers greater hypersensitivity to FR than 35S-HFR1 expression, and only seedlings expressing 35S-HFR1(ΔN) display constitutive photomorphogenesis. These phenotypic differences can be attributed to the instability of HFR1 compared with HFR1(ΔN). In transgenic plants, HFR1 levels are significantly elevated upon induced expression of a dominant-negative COP1 mutant that interferes with endogenous COP1 E3 activity. Moreover, induced expression of wild-type COP1 in transgenic plants accelerates post-translational degradation of HFR1 under FR light. Taken together, our results show that HFR1 is ubiquitinated by COP1 E3 ligase and marked for post-translational degradation during photomorphogenesis.

Different RNA species are exported from the nucleus by distinct mechanisms. Among the different RNAs, mRNAs and major spliceosomal U snRNAs share several structural similarities, yet they are exported by distinct factors. We previously showed that U1 snRNAs behaved like an mRNA in nuclear export if various ∼300-nucleotide fragments were inserted in a central position. Here we show that this export switch is dependent on the length of the insertion but independent of its position, indicating unequivocally that this switch is indeed the result of RNA length. We also show that intronless mRNAs can be progressively converted to use the U snRNA export pathway if the mRNAs are progressively shortened by deletion. In addition, immunoprecipitation experiments show that the protein composition of export RNPs is influenced by RNA length. These findings indicate that RNA length is one of the key determinants of the choice of RNA export pathway. Based on these results and previous observations, a unified model of how an RNA is committed to a specific export pathway is proposed.

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths worldwide. Here, we provide evidence that the Forkhead Box (Fox) m1b (Foxm1b or Foxm1) transcription factor is essential for the development of HCC. Conditionally deleted Foxm1b mouse hepatocytes fail to proliferate and are highly resistant to developing HCC in response to a Diethylnitrosamine (DEN)/Phenobarbital (PB) liver tumor-induction protocol. The mechanism of resistance to HCC development is associated with nuclear accumulation of the cell cycle inhibitor p27Kip1 protein and reduced expression of the Cdk1-activator Cdc25B phosphatase. We showed that the Foxm1b transcription factor is a novel inhibitory target of the p19ARF tumor suppressor. Furthermore, we demonstrated that conditional overexpression of Foxm1b protein in osteosarcoma U2OS cells greatly enhances anchorage-independent growth of cell colonies on soft agar. A p19ARF 26–44 peptide containing nine D-Arg to enhance cellular uptake of the peptide was sufficient to significantly reduce both Foxm1b transcriptional activity and Foxm1b-induced growth of U2OS cell colonies on soft agar. These results suggest that this (D-Arg)9-p19ARF 26–44 peptide is a potential therapeutic inhibitor of Foxm1b function during cellular transformation. Our studies demonstrate that the Foxm1b transcription factor is required for proliferative expansion during tumor progression and constitutes a potential new target for therapy of human HCC tumors.

Human immunodeficiency virus Rev facilitates the cytoplasmic accumulation of viral RNAs that contain a Rev binding site. A human Rev-interacting protein (hRIP) was originally identified based on its ability to interact with the Rev nuclear export signal (NES) in yeast two-hybrid assays. To date, however, the function of hRIP and a role for hRIP in Rev-directed RNA export have remained elusive. Here we ablate hRIP activity with a dominant-negative mutant or RNA interference and analyze Rev function by RNA in situ hybridization. We find, unexpectedly, that in the absence of functional hRIP, Rev-directed RNAs mislocalize and aberrantly accumulate at the nuclear periphery, where hRIP is localized. In contrast, in the absence of Rev or the Rev cofactor CRM1, Rev-directed RNAs remain nuclear. We further show that the RNA mislocalization pattern resulting from loss of hRIP activity is highly specific to Rev function: the intracellular distribution of cellular poly(A)+ mRNA, nuclear proteins, and, most important, NES-containing proteins, are unaffected. Thus, hRIP is an essential cellular Rev cofactor, which acts at a previously unanticipated step in HIV-1 RNA export: movement of RNAs from the nuclear periphery to the cytoplasm.

Nuclear matrix attachment regions (MARs) are regulatory DNA sequences that are important for higher-order chromatin organization, long-range enhancer function, and extension of chromatin modifications. Here we characterize a novel cell type-specific MAR-binding protein, SATB2, which binds to the MARs of the endogenous immunoglobulin μ locus in pre-B cells and enhances gene expression. We found that SATB2 differs from the closely related thymocyte-specific protein SATB1 by modifications of two lysines with the small ubiquitive related modifier (SUMO), which are augmented specifically by the SUMO E3 ligase PIAS1. Mutations of the SUMO conjugation sites of SATB2 enhance its activation potential and association with endogenous MARs in vivo, whereas N-terminal fusions with SUMO1 or SUMO3 decrease SATB2-mediated gene activation. Sumoylation is also involved in targeting SATB2 to the nuclear periphery, raising the possibility that this reversible modification of a MAR-binding protein may contribute to the modulation of subnuclear DNA localization.

Blood vessel formation is a complex morphological process that is only beginning to be understood at the molecular level. In this study, we demonstrate a novel and critical role for the small GTPase, RhoB, in vascular development. RhoB null mice have retarded vascular development in the retina characterized by altered sprout morphology. Moreover, pharmaceutical means to deplete RhoB in neonatal rats is associated with apoptosis in the sprouting endothelial cells of newly forming vessels. Similarly, acute depletion of RhoB by antisense or dominant-negative strategies in primary endothelial cell culture models led to apoptosis and failures in tube formation. We identified a novel link between RhoB and the Akt survival signaling pathway to explain these changes. Confocal microscopy revealed that RhoB is highly localized to the nuclear margin with a small percentage found inside the nucleus. Similarly, total Akt is throughout the cell but has increased accumulation at the nuclear margin, and active phosphorylated Akt is found primarily inside the nucleoplasm, where it partially colocalizes with the RhoB therein. We show that this colocalization is functionally relevant, because when RhoB was depleted, Akt was excluded from the nucleus and total cellular Akt protein was decreased in a proteosome-dependent manner. Because the function of RhoB in vivo appears to only be rate limiting for endothelial cell sprouting, we propose that RhoB has a novel stage-specific function to regulate endothelial cell survival during vascular development. RhoB may offer a therapeutic target in diseases such as cancer, diabetic retinopathy, and macular degeneration, where the disruption of sprouting angiogenesis would be desirable.

Mammalian CLOCK and BMAL1 are two members of bHLH-PAS-containing family of transcription factors that represent the positive elements of circadian autoregulatory feedback loop. In the form of a heterodimer, they drive transcription from E-box enhancer elements in the promoters of responsive genes. We have examined abundance, posttranslational modifications, cellular localization of endogenous and ectopically expressed CLOCK and BMAL1 proteins. Nuclear/cytoplasm distribution of CLOCK was found to be under circadian regulation. Analysis of subcellular localization of CLOCK in embryo fibroblasts of mice carrying different germ-line circadian mutations showed that circadian regulation of nuclear accumulation of CLOCK is BMAL1-dependent. Formation of CLOCK/BMAL1 complex following ectopic coexpression of both proteins is followed by their codependent phosphorylation, which is tightly coupled to CLOCK nuclear translocation and degradation. This binding-dependent coregulation is specific for CLOCK/BMAL1 interaction, as no other PAS domain protein that can form a complex with either CLOCK or BMAL1 was able to induce similar effects. Importantly, all posttranslational events described in our study are coupled with active transactivation complex formation, which argues for their significant functional role. Altogether, these results provide evidence for an additional level of circadian system control, which is based on regulation of transcriptional activity or/and availability of CLOCK/BMAL1 complex.

Spen proteins regulate the expression of key transcriptional effectors in diverse signaling pathways. They are large proteins characterized by N-terminal RNA-binding motifs and a highly conserved C-terminal SPOC domain. The specific biological role of the SPOC domain (Spenparalog andorthologC-terminal domain), and hence, the common function of Spen proteins, has been unclear to date. The Spen protein, SHARP (SMRT/HDAC1-associatedrepressorprotein), was identified as a component of transcriptional repression complexes in both nuclear receptor and Notch/RBP-Jκ signaling pathways. We have determined the 1.8 Å crystal structure of the SPOC domain from SHARP. This structure shows that essentially all of the conserved surface residues map to a positively charged patch. Structure-based mutational analysis indicates that this conserved region is responsible for the interaction between SHARP and the universal transcriptional corepressor SMRT/NCoR (silencingmediator forretinoid andthyroid receptors/nuclear receptorcorepressor. We demonstrate that this interaction involves a highly conserved acidic motif at the C terminus of SMRT/NCoR. These findings suggest that the conserved function of the SPOC domain is to mediate interaction with SMRT/NCoR corepressors, and that Spen proteins play an essential role in the repression complex.

During the earliest stages ofCaenorhabditis elegansembryogenesis, the transcription factor SKN-1 initiates development of the digestive system and other mesendodermal tissues. Postembryonic SKN-1 functions have not been elucidated. SKN-1 binds to DNA through a unique mechanism, but is distantly related to basic leucine-zipper proteins that orchestrate the major oxidative stress response in vertebrates and yeast. Here we show that despite its distinct mode of target gene recognition, SKN-1 functions similarly to resist oxidative stress inC. elegans. During postembryonic stages, SKN-1 regulates a key Phase II detoxification gene through constitutive and stress-inducible mechanisms in the ASI chemosensory neurons and intestine, respectively. SKN-1 is present in ASI nuclei under normal conditions, and accumulates in intestinal nuclei in response to oxidative stress.skn-1mutants are sensitive to oxidative stress and have shortened lifespans. SKN-1 represents a connection between developmental specification of the digestive system and one of its most basic functions, resistance to oxidative and xenobiotic stress. This oxidative stress response thus appears to be both widely conserved and ancient, suggesting that the mesendodermal specification role of SKN-1 was predated by its function in these detoxification mechanisms.

Meiosis is thought to require the protein kinase Ime2 early for DNA replication and the cyclin-dependent kinase Cdc28 late for chromosome segregation. To elucidate the roles of these kinases, we inhibited their activities early and late using conditional mutants that are sensitive to chemical inhibitors. Our studies reveal that both Cdc28 and Ime2 have critical roles in meiotic S phase and M phase. Early inhibition of analog-sensitivecdc28-as1blocked DNA replication, revealing a previously undetected role for Cdc28. Yet Cdc28 was dispensable for one of its functions in the mitotic cell cycle, degradation of Sic1. Late addition of inhibitor toime2-as1revealed unexpected roles of Ime2 in the initiation and execution of chromosome segregation. The requirement of Ime2 for M phase is partially explained by its stimulation of the key meiotic transcription factor Ndt80, which is needed in turn for high Cdc28 activity. In accordance with a late role for Ime2, we observed an increase in its activity during M phase that depended on Cdc28 and Ndt80. We speculate that several unique features of the meiotic cell division reflect a division of labor and regulatory coordination between Ime2 and Cdc28.

Genomic imprinting in mammals marks the two parental alleles resulting in differential gene expression. Imprinted loci are characterized by distinct epigenetic modifications such as differential DNA methylation and asynchronous replication timing. To determine the role of DNA methylation in replication timing of imprinted loci, we analyzed replication timing inDnmt1- andDnmt3L-deficient embryonic stem (ES) cells, which lack differential DNA methylation and imprinted gene expression. Asynchronous replication is maintained in these ES cells, indicating that asynchronous replication is parent-specific without the requirement for differential DNA methylation. Imprinting centers are required for regional control of imprinted gene expression. Analysis of replication fork movement and three-dimensional RNA and DNA fluoroscent in situ hybridization (FISH) analysis of theIgf2-H19locus in various cell types indicate that theIgf2-H19imprinting center differentially regulates replication timing of nearby replicons and subnuclear localization. Based on these observations, we suggest a model in whichciselements containing nonmethylation imprints are responsible for the movement of parental imprinted loci to distinct nuclear compartments with different replication characteristics resulting in asynchronous replication timing.

Yeast possess a plasma membrane sensor of external amino acids that functions as a ligand-activated receptor. This multimeric sensor, dubbed the SPS sensor, initiates signals that regulate the expression of genes required for proper amino acid uptake. Stp1p and Stp2p are transcription factors that bind to specific sequences within the promoters of SPS-sensor-regulated genes. These factors exhibit redundant and overlapping abilities to activate transcription. We have found that Stp1p and Stp2p are synthesized as latent cytoplasmic precursors. In response to extracellular amino acids, the SPS sensor induces the rapid endoproteolytic processing of Stp1p and Stp2p. The processing of Stp1p/Stp2p occurs independently of proteasome function and without the apparent involvement of additional components. The shorter forms of these transcription factors, lacking N-terminal inhibitory domains, are targeted to the nucleus, where they transactivate SPS-sensor target genes. These results define a completely unique and streamline metabolic control pathway that directly routes environmental signals initiated at the plasma membrane to transcriptional activation in the nucleus of yeast.

Activator-mediated transcription complex assembly on templates lacking chromatin requires the interaction of activators with two major coactivator complexes: TFIID and mediator. Here we employed immobilized template assays to correlate transcriptional activation with mediator and TFIID recruitment. In reactions reconstituted with purified proteins, we found that activator, TFIID, and mediator engage in reciprocal cooperative interactions to form a complex on promoter DNA. Preassembly of the coactivator complex accelerates the rate of transcription in a cell-free system depleted of TFIID and mediator. Our data argue that this coactivator complex is an intermediate in the assembly of an active transcription complex. Furthermore, the reciprocity of the interactions demonstrates that the complex could in principle be nucleated with either TFIID or mediator, implying that alternative pathways could be utilized to generate diversity in the way activators function in vivo.

Expansion of CAG trinucleotide repeats that encode polyglutamine is the underlying cause of at least nine inherited human neurodegenerative disorders, including Huntington's disease and spinocerebellar ataxias. PolyQ fragments accumulate as aggregates in the cytoplasm and/or in the nucleus, and induce neuronal cell death. However, the molecular mechanism of polyQ-induced cell death is controversial. Here, we show the following: (1) polyQ with pathogenic repeat length triggers ER stress through proteasomal dysfunction; (2) ER stress activates ASK 1 through formation of an IRE1–TRAF2–ASK1 complex; and (3)ASK1−/− primary neurons are defective in polyQ-, proteasome inhibitor-, and ER stress-induced JNK activation and cell death. These findings suggest that ASK1 is a key element in ER stress-induced cell death that plays an important role in the neuropathological alterations in polyQ diseases.

Sister chromatid separation at the metaphase-to-anaphase transition is induced by the proteolytic cleavage of one of the cohesin complex subunits. This process is mediated by a conserved protease called separase. Separase is associated with its inhibitor, securin, until the time of anaphase initiation, when securin is degraded in an anaphase-promoting complex/cyclosome (APC/C)-dependent manner. In budding yeast securin/Pds1 not only inhibits separase/Esp1, but also promotes its nuclear localization. The molecular mechanism and regulation of this nuclear targeting are presently unknown. Here we show that Pds1 is a substrate of the cyclin-dependent kinase Cdc28. Phosphorylation of Pds1 by Cdc28 is important for efficient binding of Pds1 to Esp1 and for promoting the nuclear localization of Esp1. Our results uncover a previously unknown mechanism for regulating the Pds1–Esp1 interaction and shed light on a novel role for Cdc28 in promoting the metaphase-to-anaphase transition in budding yeast.