Hepatic FASN deficiency differentially affects nonalcoholic fatty liver disease and diabetes in mouse obesity models.

Nonalcoholic fatty liver disease (NAFLD) and type 2 diabetes are interacting comorbidities of obesity, and increased hepatic de novo lipogenesis (DNL), driven by hyperinsulinemia and carbohydrate overload, contributes to their pathogenesis. Fatty acid synthase (FASN), a key enzyme of hepatic DNL, is upregulated in association with insulin resistance. However, the therapeutic potential of targeting FASN in hepatocytes for obesity-associated metabolic diseases is unknown. Here, we show that hepatic FASN deficiency differentially affects NAFLD and diabetes depending on the etiology of obesity. Hepatocyte-specific ablation of FASN ameliorated NAFLD and diabetes in melanocortin 4 receptor-deficient mice but not in mice with diet-induced obesity. In leptin-deficient mice, FASN ablation alleviated hepatic steatosis and improved glucose tolerance but exacerbated fed hyperglycemia and liver dysfunction. The beneficial effects of hepatic FASN deficiency on NAFLD and glucose metabolism were associated with suppression of DNL and attenuation of gluconeogenesis and fatty acid oxidation, respectively. The exacerbation of fed hyperglycemia by FASN ablation in leptin-deficient mice appeared attributable to impairment of hepatic glucose uptake triggered by glycogen accumulation and citrate-mediated inhibition of glycolysis. Further investigation of the therapeutic potential of hepatic FASN inhibition for NAFLD and diabetes in humans should thus consider the etiology of obesity.

Crystal structure of GCN5 PCAF N-terminal domain reveals atypical ubiquitin ligase structure.

General control nonderepressible 5 (GCN5, also known as Kat2a) and p300/CBP-associated factor (PCAF, also known as Kat2b) are two homologous acetyltransferases. Both proteins share similar domain architecture consisting of a PCAF N-terminal (PCAF_N) domain, acetyltransferase domain, and a bromodomain. PCAF also acts as a ubiquitin E3 ligase whose activity is attributable to the PCAF_N domain, but its structural aspects are largely unknown. Here, we demonstrated that GCN5 exhibited ubiquitination activity in a similar manner to PCAF and its activity was supported by the ubiquitin-conjugating enzyme UbcH5. Moreover, we determined the crystal structure of the PCAF_N domain at 1.8 Å resolution and found that PCAF_N domain folds into a helical structure with a characteristic binuclear zinc region, which was not predicted from sequence analyses. The zinc region is distinct from known E3 ligase structures, suggesting this region may form a new class of E3 ligase. Our biochemical and structural study provides new insight into not only the functional significance of GCN5 but also into ubiquitin biology.

PHD3 regulates glucose metabolism by suppressing stress-induced signalling and optimising gluconeogenesis and insulin signalling in hepatocytes.

Glucagon-mediated gene transcription in the liver is critical for maintaining glucose homeostasis. Promoting the induction of gluconeogenic genes and blocking that of insulin receptor substrate (Irs)2 in hepatocytes contributes to the pathogenesis of type 2 diabetes. However, the molecular mechanism by which glucagon signalling regulates hepatocyte metabolism is not fully understood. We previously showed that a fasting-inducible signalling module consisting of general control non-repressed protein 5, co-regulator cAMP response element-binding protein binding protein/p300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain 2, and protein kinase A is required for glucagon-induced transcription of gluconeogenic genes. The present study aimed to identify the downstream effectors of this module in hepatocytes by examining glucagon-induced potential target genes. One of these genes was prolyl hydroxylase domain (PHD)3, which suppressed stress signalling through inhibition of the IκB kinase-nuclear factor-κB pathway in a proline hydroxylase-independent manner to maintain insulin signalling. PHD3 was also required for peroxisome proliferator-activated receptor γ coactivator 1α-induced gluconeogenesis, which was dependent on proline hydroxylase activity, suggesting that PHD3 regulates metabolism in response to glucagon as well as insulin. These findings demonstrate that glucagon-inducible PHD3 regulates glucose metabolism by suppressing stress signalling and optimising gluconeogenesis and insulin signalling in hepatocytes.

CACUL1/CAC1 attenuates p53 activity through PML post-translational modification.

Promyelocytic leukaemia (PML) is a tumor suppressor protein covalently conjugated with SUMO family proteins, leading to the formation of PML nuclear bodies (NBs). PML-NBs provide a platform for efficient posttranslational modification of targets and protein-protein interaction, contributing to the adjustment of gene expression and chromatin integrity. Although PML SUMOylation is thought to play important roles in diverse cellular functions, the control mechanisms of adequate modification levels have remained unsolved. Here, we report that Cullin-related protein CACUL1/CAC1 (CACUL1) inhibits PML posttranslational modification. CACUL1 interacts with PML and suppresses PML SUMOylation, leading to the regulation of PML-NB size in the nucleus. We also found that Ubc9, a SUMO-conjugating enzyme, binds to CACUL1 and antagonizes the interaction between CACUL1 and PML. Furthermore, CACUL1 attenuates p53 transcriptional activity. These data suggest that CACUL1 is a novel regulator that negatively controls p53 activity through the regulation of PML SUMOylation.

SWI/SNF chromatin-remodeling complexes function in noncoding RNA-dependent assembly of nuclear bodies.

Paraspeckles are subnuclear structures that form around nuclear paraspeckle assembly transcript 1 (NEAT1) long noncoding RNA (lncRNA). Recently, paraspeckles were shown to be functional nuclear bodies involved in stress responses and the development of specific organs. Paraspeckle formation is initiated by transcription of the NEAT1 chromosomal locus and proceeds in conjunction with NEAT1 lncRNA biogenesis and a subsequent assembly step involving >40 paraspeckle proteins (PSPs). In this study, subunits of SWItch/Sucrose NonFermentable (SWI/SNF) chromatin-remodeling complexes were identified as paraspeckle components that interact with PSPs and NEAT1 lncRNA. EM observations revealed that SWI/SNF complexes were enriched in paraspeckle subdomains depleted of chromatin. Knockdown of SWI/SNF components resulted in paraspeckle disintegration, but mutation of the ATPase domain of the catalytic subunit BRG1 did not affect paraspeckle integrity, indicating that the essential role of SWI/SNF complexes in paraspeckle formation does not require their canonical activity. Knockdown of SWI/SNF complexes barely affected the levels of known essential paraspeckle components, but markedly diminished the interactions between essential PSPs, suggesting that SWI/SNF complexes facilitate organization of the PSP interaction network required for intact paraspeckle assembly. The interactions between SWI/SNF components and essential PSPs were maintained in NEAT1-depleted cells, suggesting that SWI/SNF complexes not only facilitate interactions between PSPs, but also recruit PSPs during paraspeckle assembly. SWI/SNF complexes were also required for Satellite III lncRNA-dependent formation of nuclear stress bodies under heat-shock conditions. Our data suggest the existence of a common mechanism underlying the formation of lncRNA-dependent nuclear body architectures in mammalian cells.

NEAT1 long noncoding RNA regulates transcription via protein sequestration within subnuclear bodies.

Paraspeckles are subnuclear structures formed around nuclear paraspeckle assembly transcript 1 (NEAT1)/MENε/ß long noncoding RNA (lncRNA). Here we show that paraspeckles become dramatically enlarged after proteasome inhibition. This enlargement is mainly caused by NEAT1 transcriptional up-regulation rather than accumulation of undegraded paraspeckle proteins. Of interest, however, using immuno-electron microscopy, we find that key paraspeckle proteins become effectively depleted from the nucleoplasm by 50% when paraspeckle assembly is enhanced, suggesting a sequestration mechanism. We also perform microarrays from NEAT1-knockdown cells and find that NEAT1 represses transcription of several genes, including the RNA-specific adenosine deaminase B2 (ADARB2) gene. In contrast, the NEAT1-binding paraspeckle protein splicing factor proline/glutamine-rich (SFPQ) is required for ADARB2 transcription. This leads us to hypothesize that ADARB2 expression is controlled by NEAT1-dependent sequestration of SFPQ. Accordingly, we find that ADARB2 expression is strongly reduced upon enhanced SFPQ sequestration by proteasome inhibition, with concomitant reduction in SFPQ binding to the ADARB2 promoter. Finally, NEAT1(-/-) fibroblasts are more sensitive to proteasome inhibition, which triggers cell death, suggesting that paraspeckles/NEAT1 attenuates the cell death pathway. These data further confirm that paraspeckles are stress-responsive nuclear bodies and provide a model in which induced NEAT1 controls target gene transcription by protein sequestration into paraspeckles.

Paraspeckle formation during the biogenesis of long non-coding RNAs.

Paraspeckles are unique subnuclear structures that are built around a specific long non-coding RNA (lncRNA), NEAT1, which is comprised of two isoforms (NEAT1_1 and NEAT1_2) that are produced by alternative 3'-end processing. NEAT1 lncRNAs are unusual RNA polymerase II transcripts that lack introns. The non-polyadenylated 3'-end of NEAT1_2 is non-canonically processed by RNase P. NEAT1_2 is an essential component for paraspeckle formation. Paraspeckles form during the NEAT1_2 lncRNA biogenesis process, which encompasses transcription from its own chromosome locus through lncRNA processing and accumulation. Recent RNAi analyses of 40 paraspeckle proteins (PSPs) identified four PSPs that are required for paraspeckle formation by mediating NEAT1 processing and accumulation. In particular, HNRNPK was shown to arrest CFIm-dependent NEAT1_1 polyadenylation, leading to NEAT1_2 synthesis. The other three PSPs were required for paraspeckle formation, but did not affect NEAT1_2 expression. This observation suggests that NEAT1_2 accumulation is necessary but not sufficient for paraspeckle formation. An additional step, presumably the bundling of NEAT1 ribonucleoprotein sub-complexes, may be required for construction of the intact paraspeckle structure. NEAT1 expression is likely regulated at transcriptional and post-transcriptional steps under certain stress conditions, suggesting roles for paraspeckles in the lncRNA-mediated regulation of gene expression, such as the nucleocytoplasmic transport of mRNA in response to certain stimuli.

Alternative 3'-end processing of long noncoding RNA initiates construction of nuclear paraspeckles.

Paraspeckles are unique subnuclear structures built around a specific long noncoding RNA, NEAT1, which is comprised of two isoforms produced by alternative 3'-end processing (NEAT1_1 and NEAT1_2). To address the precise molecular processes that lead to paraspeckle formation, we identified 35 paraspeckle proteins (PSPs), mainly by colocalization screening with a fluorescent protein-tagged full-length cDNA library. Most of the newly identified PSPs possessed various putative RNA-binding domains. Subsequent RNAi analyses identified seven essential PSPs for paraspeckle formation. One of the essential PSPs, HNRNPK, appeared to affect the production of the essential NEAT1_2 isoform by negatively regulating the 3'-end polyadenylation of the NEAT1_1 isoform. An in vitro 3'-end processing assay revealed that HNRNPK arrested binding of the CPSF6-NUDT21 (CFIm) complex in the vicinity of the alternative polyadenylation site of NEAT1_1. In vitro binding assays showed that HNRNPK competed with CPSF6 for binding to NUDT21, which was the underlying mechanism to arrest CFIm binding by HNRNPK. This HNRNPK function led to the preferential accumulation of NEAT1_2 and initiated paraspeckle construction with multiple PSPs.

U7 small nuclear ribonucleoprotein represses histone gene transcription in cell cycle-arrested cells.

Histone gene expression is tightly coordinated with DNA replication, as it is activated at the onset of S phase and suppressed at the end of S phase. Replication-dependent histone gene expression is precisely controlled at both transcriptional and posttranscriptional levels. U7 small nuclear ribonucleoprotein (U7 snRNP) is involved in the 3'-end processing of nonpolyadenylated histone mRNAs, which is required for S phase-specific gene expression. The present study reports a unique function of U7 snRNP in the repression of histone gene transcription under cell cycle-arrested conditions. Elimination of U7 snRNA with an antisense oligonucleotide in HeLa cells as well as in nontransformed human lung fibroblasts resulted in elevated levels of replication-dependent H1, H2A, H2B, H3, and H4 histone mRNAs but not of replication-independent H3F3B histone mRNA. An analogous effect was observed upon depletion of Lsm10, a component of the U7 snRNP-specific Sm ring, with siRNA. Pulse-chase experiments revealed that U7 snRNP acts to repress transcription without remarkably altering mRNA stability. Mass spectrometric analysis of the captured U7 snRNP from HeLa cell extracts identified heterogeneous nuclear (hn)RNP UL1 as a U7 snRNP interaction partner. Further knockdown and overexpression experiments revealed that hnRNP UL1 is responsible for U7 snRNP-dependent transcriptional repression of replication-dependent histone genes. Chromatin immunoprecipitation confirmed that hnRNP UL1 is recruited to the histone gene locus only when U7 snRNP is present. These findings support a unique mechanism of snRNP-mediated transcriptional control that restricts histone synthesis to S phase, thereby preventing the potentially toxic effects of histone synthesis at other times in the cell cycle.

Archaeal ribosomal stalk protein interacts with translation factors in a nucleotide-independent manner via its conserved C terminus.

Protein synthesis on the ribosome requires translational GTPase factors to bind to the ribosome in the GTP-bound form, take individual actions that are coupled with GTP hydrolysis, and dissociate, usually in the GDP-bound form. The multiple copies of the flexible ribosomal stalk protein play an important role in these processes. Using biochemical approaches and the stalk protein from a hyperthermophilic archaeon, Pyrococcus horikoshii, we here provide evidence that the conserved C terminus of the stalk protein aP1 binds directly to domain I of the elongation factor aEF-2, irrespective of whether aEF-2 is bound to GTP or GDP. Site-directed mutagenesis revealed that four hydrophobic amino acids at the C terminus of aP1, Leu-100, 103, 106, and Phe-107, are crucial for the direct binding. P1 was also found to bind to the initiation factor aIF5B, as well as aEF-1α, but not aIF2γ, via its C terminus. Moreover, analytical ultracentrifugation and gel mobility shift analyses showed that a heptameric complex of aP1 and aP0, aP0(aP1)(2)(aP1)(2)(aP1)(2), can bind multiple aEF-2 molecules simultaneously, which suggests that individual copies of the stalk protein are accessible to the factor. The functional significance of the C terminus of the stalk protein was also shown using the eukaryotic proteins P1/P2 and P0. It is likely that the conserved C terminus of the stalk proteins of archaea and eukaryotes can bind to translation factors both before and after GTP hydrolysis. This consistent binding ability of the stalk protein may contribute to maintaining high concentrations of translation factors around the ribosome, thus promoting translational efficiency.

Paraspeckles are subpopulation-specific nuclear bodies that are not essential in mice.

Nuclei of higher organisms are well structured and have multiple, distinct nuclear compartments or nuclear bodies. Paraspeckles are recently identified mammal-specific nuclear bodies ubiquitously found in most cells cultured in vitro. To investigate the physiological role of paraspeckles, we examined the in vivo expression patterns of two long noncoding RNAs, NEAT1_1 and NEAT1_2, which are essential for the architectural integrity of nuclear bodies. Unexpectedly, these genes were only strongly expressed in a particular subpopulation of cells in adult mouse tissues, and prominent paraspeckle formation was observed only in the cells highly expressing NEAT1_2. To further investigate the cellular functions of paraspeckles, we created an animal model lacking NEAT1 by gene targeting. These knockout mice were viable and fertile under laboratory growth conditions, showing no apparent phenotypes except for the disappearance of paraspeckles. We propose that paraspeckles are nonessential, subpopulation-specific nuclear bodies formed secondary to particular environmental triggers.

Structural basis for translation factor recruitment to the eukaryotic/archaeal ribosomes.

The archaeal ribosomal stalk complex has been shown to have an apparently conserved functional structure with eukaryotic pentameric stalk complex; it provides access to eukaryotic elongation factors at levels comparable to that of the eukaryotic stalk. The crystal structure of the archaeal heptameric (P0(P1)(2)(P1)(2)(P1)(2)) stalk complex shows that the rRNA anchor protein P0 consists of an N-terminal rRNA-anchoring domain followed by three separated spine helices on which three P1 dimers bind. Based on the structure, we have generated P0 mutants depleted of any binding site(s) for P1 dimer(s). Factor-dependent GTPase assay of such mutants suggested that the first P1 dimer has higher activity than the others. Furthermore, we constructed a model of the archaeal 50 S with stalk complex by superposing the rRNA-anchoring domain of P0 on the archaeal 50 S. This model indicates that the C termini of P1 dimers where translation factors bind are all localized to the region between the stalk base of the 50 S and P0 spine helices. Together with the mutational experiments we infer that the functional significance of multiple copies of P1 is in creating a factor pool within a limited space near the stalk base of the ribosome.

Three binding sites for stalk protein dimers are generally present in ribosomes from archaeal organism.

Ribosomes have a characteristic protuberance termed the stalk, which is indispensable for ribosomal function. The ribosomal stalk has long been believed to be a pentameric protein complex composed of two sets of protein dimers, L12-L12, bound to a single anchor protein, although ribosomes carrying three L12 dimers were recently discovered in a few thermophilic bacteria. Here we have characterized the stalk complex from Pyrococcus horikoshii, a thermophilic species of Archaea. This complex is known to be composed of proteins homologous to eukaryotic counterparts rather than bacterial ones. In truncation experiments of the C-terminal regions of the anchor protein Ph-P0, we surprisingly observed three Ph-L12 dimers bound to the C-terminal half of Ph-P0, and the binding site for the third dimer was unique to the archaeal homologs. The stoichiometry of the heptameric complex Ph-P0(Ph-L12)(2)(Ph-L12)(2)(Ph-L12)(2) was confirmed by mass spectrometry of the intact complex. In functional tests, ribosomes carrying a single Ph-L12 dimer had significant activity, but the addition of the second and third dimers increased the activity. A bioinformatics analysis revealed the evidence that ribosomes from all archaeal and also from many bacterial organisms may contain a heptameric complex at the stalk, whereas eukaryotic ribosomes seem to contain exclusively a pentameric stalk complex, thus modifying our view of the stalk structure significantly.

The N-terminal regions of eukaryotic acidic phosphoproteins P1 and P2 are crucial for heterodimerization and assembly into the ribosomal GTPase-associated center.

Acidic phosphoproteins P1 and P2 form a heterodimer and play a crucial role in assembly of the GTPase-associated center in eukaryotic ribosomes and in ribosomal interaction with translation factors. We investigated the structural elements within P1 and P2 essential for their dimerization and for ribosomal function. Truncation of the N-terminal 10 amino acids in either P1 or P2 and swapping of the N-terminal 10 amino acid sequences between these two proteins disrupted their dimerization, binding to P0 and P0 binding to rRNA. In contrast, truncation of the C-terminal halves of P1 and P2 as well as swapping of these parts between them gave no significant effects. The protein dimers containing the C-terminal truncation mutants or swapped variants were assembled with P0 onto Escherichia coli 50 S subunits deficient in the homologous protein L10 and L7/L12 and gave reduced ribosomal activity in terms of eukaryotic elongation factor dependent GTPase activity and polyphenylalanine synthesis. The results indicate that the N-terminal 10 amino acid sequences of both P1 and P2 are crucial for P1-P2 heterodimerization and for their functional assembly with P0 into the GTPase-associated center, whereas the C-terminal halves of P1 and P2 are not essential for the assembly.

In vitro reconstitution of the GTPase-associated centre of the archaebacterial ribosome: the functional features observed in a hybrid form with Escherichia coli 50S subunits.

We cloned the genes encoding the ribosomal proteins Ph (Pyrococcus horikoshii)-P0, Ph-L12 and Ph-L11, which constitute the GTPase-associated centre of the archaebacterium Pyrococcus horikoshii. These proteins are homologues of the eukaryotic P0, P1/P2 and eL12 proteins, and correspond to Escherichia coli L10, L7/L12 and L11 proteins respectively. The proteins and the truncation mutants of Ph-P0 were overexpressed in E. coli cells and used for in vitro assembly on to the conserved domain around position 1070 of 23S rRNA (E. coli numbering). Ph-L12 tightly associated as a homodimer and bound to the C-terminal half of Ph-P0. The Ph-P0.Ph-L12 complex and Ph-L11 bound to the 1070 rRNA fragments from the three biological kingdoms in the same manner as the equivalent proteins of eukaryotic and eubacterial ribosomes. The Ph-P0.Ph-L12 complex and Ph-L11 could replace L10.L7/L12 and L11 respectively, on the E. coli 50S subunit in vitro. The resultant hybrid ribosome was accessible for eukaryotic, as well as archaebacterial elongation factors, but not for prokaryotic elongation factors. The GTPase and polyphenylalanine-synthetic activity that is dependent on eukaryotic elongation factors was comparable with that of the hybrid ribosomes carrying the eukaryotic ribosomal proteins. The results suggest that the archaebacterial proteins, including the Ph-L12 homodimer, are functionally accessible to eukaryotic translation factors.

A mode of assembly of P0, P1, and P2 proteins at the GTPase-associated center in animal ribosome: in vitro analyses with P0 truncation mutants.

Ribosomal P0, P1, and P2 proteins, together with the conserved domain of 28 S rRNA, constitute a major part of the GTPase-associated center in eukaryotic ribosomes. We investigated the mode of assembly in vitro by using various truncation mutants of silkworm P0. When compared with wild type (WT)-P0, the C-terminal truncation mutants CDelta65 and CDelta81 showed markedly reduced binding ability to P1 and P2, which was offset by the addition of an rRNA fragment covering the P0.P1-P2 binding site. The mutant CDelta107 lost the P1/P2 binding activity, whereas it retained the rRNA binding. In contrast, the N-terminal truncation mutants NDelta21-NDelta92 completely lost the rRNA binding, although they retained P1/P2 binding capability, implying an essential role of the N terminus of P0 for rRNA binding. The P0 mutants NDelta6, NDelta14, and CDelta18-CDelta81, together with P1/P2 and eL12, bound to the Escherichia coli core 50 S subunits deficient in L10.L7/L12 complex and L11. Analysis of incorporation of (32)P-labeled P1/P2 into the 50 S subunits with WT-P0 and CDelta81 by sedimentation analysis indicated that WT-P0 bound two copies of P1 and P2, but CDelta81 bound only one copy each. The hybrid ribosome with CDelta81 that appears to contain one P1-P2 heterodimer retained lower but considerable activities dependent on eukaryotic elongation factors. These results suggested that two P1-P2 dimers bind to close but separate regions on the C-terminal half of P0. The results were further confirmed by binding experiments using chimeric P0 mutants in which the C-terminal 81 or 107 amino acids were replaced with the homologous sequences of the archaebacterial P0.

Baculovirus-mediated expression and isolation of human ribosomal phosphoprotein P0 carrying a GST-tag in a functional state.

We constructed an overexpression system for human ribosomal phosphoprotein P0, together with P1 and P2, which is crucially important for translation. Genes for these proteins, fused with the glutathione S-transferase (GST)-tag at the N-terminus, were inserted into baculovirus and introduced to insect cells. The fusion proteins, but not the proteins without the tag, were efficiently expressed into cells as soluble forms. The fusion protein GST.P0 as well as GST.P1/GST.P2 was phosphorylated in cells as detected by incorporation of (32)P and reactivity with monoclonal anti-phosphoserine antibody. GST.P0 expressed in insect cells, but not the protein obtained in Escherichia coli, had the ability to form a complex with P1 and P2 proteins and to bind to 28S rRNA. Moreover, the GST.P0-P1-P2 complex participated in high eEF-2-dependent GTPase activity. Baculovirus expression systems appear to provide recombinant human P0 samples that can be used for studies on the structure and function.