RÉSUMÉ
When mRNAs have been transcribed and processed in the nucleus, they are exported to the cytoplasm for translation. This export is mediated by the export receptor heterodimer Mex67-Mtr2 in the yeast Saccharomyces cerevisiae (TAP-p15 in humans)1,2. Interestingly, many long non-coding RNAs (lncRNAs) also leave the nucleus but it is currently unclear why they move to the cytoplasm3. Here we show that antisense RNAs (asRNAs) accelerate mRNA export by annealing with their sense counterparts through the helicase Dbp2. These double-stranded RNAs (dsRNAs) dominate export compared with single-stranded RNAs (ssRNAs) because they have a higher capacity and affinity for the export receptor Mex67. In this way, asRNAs boost gene expression, which is beneficial for cells. This is particularly important when the expression program changes. Consequently, the degradation of dsRNA, or the prevention of its formation, is toxic for cells. This mechanism illuminates the general cellular occurrence of asRNAs and explains their nuclear export.
Sujet(s)
Transport nucléaire actif , Noyau de la cellule , Régulation de l'expression des gènes fongiques , Transport des ARN , ARN antisens , ARN double brin , ARN messager , Saccharomyces cerevisiae , Noyau de la cellule/métabolisme , Cytoplasme/métabolisme , DEAD-box RNA helicases/métabolisme , DEAD-box RNA helicases/génétique , Transporteurs nucléocytoplasmiques/métabolisme , Transporteurs nucléocytoplasmiques/génétique , ARN antisens/métabolisme , ARN antisens/génétique , ARN double brin/métabolisme , ARN double brin/génétique , ARN messager/métabolisme , ARN messager/génétique , Protéines de liaison à l'ARN/métabolisme , Protéines de liaison à l'ARN/génétique , Saccharomyces cerevisiae/génétique , Saccharomyces cerevisiae/métabolisme , Protéines de Saccharomyces cerevisiae/métabolisme , Protéines de Saccharomyces cerevisiae/génétiqueRÉSUMÉ
The nonstructural protein 1 (Nsp1) of SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) is a virulence factor that targets multiple cellular pathways to inhibit host gene expression and antiviral response. However, the underlying mechanisms of the various Nsp1-mediated functions and their contributions to SARS-CoV-2 virulence remain unclear. Among the targets of Nsp1 is the mRNA (messenger ribonucleic acid) export receptor NXF1-NXT1, which mediates nuclear export of mRNAs from the nucleus to the cytoplasm. Based on Nsp1 crystal structure, we generated mutants on Nsp1 surfaces and identified an acidic N-terminal patch that is critical for interaction with NXF1-NXT1. Photoactivatable Nsp1 probe reveals the RNA Recognition Motif (RRM) domain of NXF1 as an Nsp1 N-terminal binding site. By mutating the Nsp1 N-terminal acidic patch, we identified a separation-of-function mutant of Nsp1 that retains its translation inhibitory function but substantially loses its interaction with NXF1 and reverts Nsp1-mediated mRNA export inhibition. We then generated a recombinant (r)SARS-CoV-2 mutant on the Nsp1 N-terminal acidic patch and found that this surface is key to promote NXF1 binding and inhibition of host mRNA nuclear export, viral replication, and pathogenicity in vivo. Thus, these findings provide a mechanistic understanding of Nsp1-mediated mRNA export inhibition and establish the importance of this pathway in the virulence of SARS-CoV-2.
Sujet(s)
Transport nucléaire actif , COVID-19 , Transporteurs nucléocytoplasmiques , ARN messager , Protéines de liaison à l'ARN , SARS-CoV-2 , Protéines virales non structurales , Humains , SARS-CoV-2/métabolisme , SARS-CoV-2/pathogénicité , SARS-CoV-2/génétique , Protéines virales non structurales/métabolisme , Protéines virales non structurales/génétique , Protéines virales non structurales/composition chimique , ARN messager/génétique , ARN messager/métabolisme , Transporteurs nucléocytoplasmiques/métabolisme , Transporteurs nucléocytoplasmiques/génétique , Animaux , COVID-19/virologie , COVID-19/métabolisme , Protéines de liaison à l'ARN/métabolisme , Protéines de liaison à l'ARN/génétique , Réplication virale , Noyau de la cellule/métabolisme , Cellules Vero , Virulence , Chlorocebus aethiops , Cellules HEK293RÉSUMÉ
mRNAs continually change their protein partners throughout their lifetimes, yet our understanding of mRNA-protein complex (mRNP) remodeling is limited by a lack of temporal data. Here, we present time-resolved mRNA interactome data by performing pulse metabolic labeling with photoactivatable ribonucleoside in human cells, UVA crosslinking, poly(A)+ RNA isolation, and mass spectrometry. This longitudinal approach allowed the quantification of over 700 RNA binding proteins (RBPs) across ten time points. Overall, the sequential order of mRNA binding aligns well with known functions, subcellular locations, and molecular interactions. However, we also observed RBPs with unexpected dynamics: the transcription-export (TREX) complex recruited posttranscriptionally after nuclear export factor 1 (NXF1) binding, challenging the current view of transcription-coupled mRNA export, and stress granule proteins prevalent in aged mRNPs, indicating roles in late stages of the mRNA life cycle. To systematically identify mRBPs with unknown functions, we employed machine learning to compare mRNA binding dynamics with Gene Ontology (GO) annotations. Our data can be explored at chronology.rna.snu.ac.kr.
Sujet(s)
ARN messager , Protéines de liaison à l'ARN , Humains , ARN messager/métabolisme , ARN messager/génétique , Protéines de liaison à l'ARN/métabolisme , Protéines de liaison à l'ARN/génétique , Ribonucléoprotéines/métabolisme , Ribonucléoprotéines/génétique , Liaison aux protéines , Transporteurs nucléocytoplasmiques/métabolisme , Transporteurs nucléocytoplasmiques/génétique , Cellules HeLa , Facteurs temps , Apprentissage machineRÉSUMÉ
Regulating nuclear export precisely is essential for maintaining mRNA homeostasis and impacts tumor progression. However, the mechanisms governing nuclear mRNA export remain poorly elucidated. Herein, it is revealed that the enhanced hypoxic long no-ncoding RNA (lncRNA prostate cancer associated transcript 6 (PCAT6) in breast cancer (BC) promotes the nuclear export of m6A-modified mRNAs, bolstering breast cancer stem cells (BCSCs) stemness and doxorubicin resistance. Clinically, hypoxic PCAT6 correlates with malignant BC features and poor prognosis. Mechanically, PCAT6 functions as a scaffold between interferon-stimulated gene 15 (ISG15) and heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2B1), leading to ISGylation of hnRNPA2B1, thus protecting hnRNPA2B1 from ubiquitination-mediated proteasomal degradation. Interestingly, as an m6A reader, hnRNPA2B1 selectively mediates m6A-tagged mRNAs nuclear export via the Aly/REF export factor (ALYREF)/ nuclear RNA export factor 1 (NXF1) complex, which promotes stemness-related genes expression. HnRNPA2B1 knockdown or mRNA export inhibition can result in the retention of nuclear m6A-tagged mRNA associated with stemness maintenance, which suppresses BCSCs self-renewal and effectively improves the efficacy of doxorubicin therapy. These findings demonstrate the pivotal role of m6A-modified mRNA nuclear export in BC progression, highlighting that the inhibition of m6A-tagged mRNA and its nuclear export is a potential therapeutic strategy for the amelioration of cancer chemotherapy.
Sujet(s)
Tumeurs du sein , Ribonucléoprotéine nucléaire hétérogène du groupe A-B , ARN messager , Humains , Tumeurs du sein/génétique , Tumeurs du sein/métabolisme , Ribonucléoprotéine nucléaire hétérogène du groupe A-B/métabolisme , Ribonucléoprotéine nucléaire hétérogène du groupe A-B/génétique , Femelle , ARN messager/génétique , ARN messager/métabolisme , Lignée cellulaire tumorale , Souris , ARN long non codant/génétique , ARN long non codant/métabolisme , Protéines de liaison à l'ARN/métabolisme , Protéines de liaison à l'ARN/génétique , Animaux , Transporteurs nucléocytoplasmiques/métabolisme , Transporteurs nucléocytoplasmiques/génétique , Cellules souches tumorales/métabolismeRÉSUMÉ
The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) accessory protein Orf6 works as an interferon antagonist, in part, by inhibiting the nuclear import activated p-STAT1, an activator of interferon-stimulated genes, and the export of the poly(A) RNA. Insight into the transport regulatory function of Orf6 has come from the observation that Orf6 binds to the nuclear pore complex (NPC) components: Rae1 and Nup98. To gain further insight into the mechanism of Orf6-mediated transport inhibition, we examined the role of Rae1 and Nup98. We show that Rae1 alone is not necessary to support p-STAT1 import or nuclear export of poly(A) RNA. Moreover, the loss of Rae1 suppresses the transport inhibitory activity of Orf6. We propose that the Rae1/Nup98 complex strategically positions Orf6 within the NPC where it alters FG-Nup interactions and their ability to support nuclear transport. In addition, we show that Rae1 is required for normal viral protein production during SARS-CoV-2 infection presumably through its role in supporting Orf6 function.
Sujet(s)
Transport nucléaire actif , COVID-19 , Pore nucléaire , Transporteurs nucléocytoplasmiques , SARS-CoV-2 , Humains , COVID-19/métabolisme , Interférons/métabolisme , Pore nucléaire/métabolisme , Complexe protéique du pore nucléaire/métabolisme , Transporteurs nucléocytoplasmiques/métabolisme , ARN messager/métabolisme , SARS-CoV-2/métabolisme , Protéines virales/métabolisme , Protéines associées à la matrice nucléaire/métabolismeRÉSUMÉ
AIMS: The purpose of this study was to explore the role of RAE1 in the invasion and metastasis of gastric cancer (GC) cells. MATERIALS AND METHODS: RAE1 expression in GC cells was determined by reverse-transcription polymerase chain reaction (qRT-PCR) and Western blotting (WB). Cell models featuring RAE1 gene silencing and overexpression were constructed by lentiviral transfection; The proliferation, migration, and invasion ability of cells were detected by cell counting, colony formation assay, would healing assay, and transwell invasion and migration test. WB analysis of ERK/MAPK signaling pathway (ERK1/2, p-ERK1/2, c-Myc) and EMT-related molecules (ZEB1, E-cadherin, N-cadherin, and Vimentin). RESULTS: The expression level of RAE1 in GC was notably higher than in adjacent tissues. Elevated RAE1 expression correlated with an unfavorable prognosis for GC patients. Knockdown of RAE1, as compared to the control group, resulted in a significant inhibition of proliferation, migration, and invasion abilities in GC cell lines. Furthermore, RAE1 knockdown led to a substantial decrease in the expression of N-cadherin, vimentin, ZEB1, p-ERK1/2, and c-Myc proteins, coupled with a marked increase in E-cadherin expression. The biological effects of RAE1 in GC cells were effectively reversed by the inhibition of the ERK/MAPK signaling pathway using SCH772984. Additionally, RAE1 knockdown demonstrated a suppressive effect on GC tumor size in vivo. Immunohistochemistry (IHC) results revealed significantly lower expression of Ki-67 in RAE1 knockout mice compared to the control group. CONCLUSIONS: RAE1 promotes GC cell migration and invasion through the ERK/MAPK pathway and is a potential therapeutic target for GC therapy.
Sujet(s)
Transition épithélio-mésenchymateuse , Tumeurs de l'estomac , Animaux , Humains , Souris , Cadhérines/génétique , Cadhérines/métabolisme , Carcinogenèse , Lignée cellulaire tumorale , Mouvement cellulaire , Prolifération cellulaire , Régulation de l'expression des gènes tumoraux , Invasion tumorale/génétique , Protéines associées à la matrice nucléaire/génétique , Protéines associées à la matrice nucléaire/métabolisme , Transporteurs nucléocytoplasmiques/génétique , Transporteurs nucléocytoplasmiques/métabolisme , Tumeurs de l'estomac/métabolisme , Tumeurs de l'estomac/anatomopathologie , Vimentine/génétique , Vimentine/métabolismeRÉSUMÉ
Nuclear mRNA export via nuclear pore complexes is an essential step in eukaryotic gene expression. Although factors involved in mRNA transport have been characterized, a comprehensive mechanistic understanding of this process and its regulation is lacking. Here, we use single-RNA imaging in yeast to show that cells use mRNA retention to control mRNA export during stress. We demonstrate that, upon glucose withdrawal, the essential RNA-binding factor Nab2 forms RNA-dependent condensate-like structures in the nucleus. This coincides with a reduced abundance of the DEAD-box ATPase Dbp5 at the nuclear pore. Depleting Dbp5, and consequently blocking mRNA export, is necessary and sufficient to trigger Nab2 condensation. The state of Nab2 condensation influences the extent of nuclear mRNA accumulation and can be recapitulated in vitro, where Nab2 forms RNA-dependent liquid droplets. We hypothesize that cells use condensation to regulate mRNA export and control gene expression during stress.
Sujet(s)
Complexe protéique du pore nucléaire , Protéines de Saccharomyces cerevisiae , Transport nucléaire actif , Noyau de la cellule/métabolisme , DEAD-box RNA helicases/métabolisme , Complexe protéique du pore nucléaire/métabolisme , Transporteurs nucléocytoplasmiques/génétique , Transporteurs nucléocytoplasmiques/métabolisme , ARN/métabolisme , ARN messager/génétique , ARN messager/métabolisme , Protéines de liaison à l'ARN/génétique , Protéines de liaison à l'ARN/métabolisme , Saccharomyces cerevisiae/génétique , Saccharomyces cerevisiae/métabolisme , Protéines de Saccharomyces cerevisiae/métabolismeRÉSUMÉ
The contact inhibition of proliferation (CIP) denotes the cell density-dependent inhibition of growth, and the loss of CIP represents a hallmark of cancer. However, the mechanism by which CIP regulates gene expression remains poorly understood. Chromatin is a highly complex structure consisting of DNA, histones, and trans-acting factors (TAFs). The binding of TAF proteins to specific chromosomal loci regulates gene expression. Therefore, profiling chromatin is crucial for gaining insight into the gene expression mechanism of CIP. In this study, using modified proteomics of TAFs bound to DNA, we identified a protein that shuttles between the nucleus and cytosol in a cell density-dependent manner. We identified TIPARP, PTGES3, CBFB, and SMAD4 as cell density-dependent nucleocytoplasmic shuttling proteins. In low-density cells, these proteins predominantly reside in the nucleus; however, upon reaching high density, they relocate to the cytosol. Given their established roles in gene regulation, our findings propose their involvement as CIP-dependent TAFs. We also identified and characterized potential open chromatin regions sensitive to changes in cell density. These findings provide insights into the modulation of chromatin structure by CIP.
Sujet(s)
Noyau de la cellule , Chromatine , Chromatine/génétique , Chromatine/métabolisme , Noyau de la cellule/métabolisme , Cytoplasme/métabolisme , Transactivateurs/métabolisme , ADN/métabolisme , Transporteurs nucléocytoplasmiques/métabolisme , Numération cellulaireRÉSUMÉ
Gle1 regulates gene expression at multiple steps from transcription to mRNA export to translation under stressed and non-stressed conditions. To better understand Gle1 function in stressed human cells, specific antibodies were generated that recognized the phosphorylation of threonine residue 102 (T102) in Gle1. A series of in vitro kinase assays indicated that T102 phosphorylation serves as a priming event for further phosphorylation in Gle1's N-terminal low complexity cluster. Indirect immunofluorescence microscopy with the anti-Gle1-pT102 antibodies revealed that basally phosphorylated Gle1 was pre-dominantly nuclear with punctate distribution; however, under sodium arsenite-induced stress, more cytoplasmic localization was detected. Immunoprecipitation with the anti-Gle1-pT102 antibody resulted in co-isolation of Gle1-pT102 with the DEAD-box protein DDX1 in a phosphatase sensitive manner. This suggested Gle1 phosphorylation might be linked to its role in regulating DDX1 during transcription termination. Notably, whereas the total Gle1-DDX1 association was decreased when Gle1 nucleocytoplasmic shuttling was disrupted, co-isolation of Gle1-pT102 and DDX1 increased under the same conditions. Taken together, these studies demonstrated that Gle1 phosphorylation impacts its cellular distribution and potentially drives nuclear Gle1 functions in transcription termination. We propose a model wherein phosphorylation of Gle1 either reduces its nucleocytoplasmic shuttling capacity or increases its binding affinity with nuclear interaction partners.
Sujet(s)
Complexe protéique du pore nucléaire , Humains , DEAD-box RNA helicases/génétique , DEAD-box RNA helicases/métabolisme , Complexe protéique du pore nucléaire/composition chimique , Complexe protéique du pore nucléaire/génétique , Complexe protéique du pore nucléaire/métabolisme , Transporteurs nucléocytoplasmiques/génétique , Transporteurs nucléocytoplasmiques/métabolisme , Phosphorylation , Noyau de la cellule/métabolismeRÉSUMÉ
Triple-negative breast cancer (TNBC) is the most fatal subtype of breast cancer; however, effective treatment strategies for TNBC are lacking. Therefore, it is important to explore the mechanism of TNBC metastasis and identify its therapeutic targets. Dysregulation of ETHE1 leads to ethylmalonic encephalopathy in humans; however, the role of ETHE1 in TNBC remains elusive. Stable cell lines with ETHE1 overexpression or knockdown were constructed to explore the biological functions of ETHE1 during TNBC progression in vitro and in vivo. Mass spectrometry was used to analyze the molecular mechanism through which ETHE1 functions in TNBC progression. ETHE1 had no impact on TNBC cell proliferation and xenograft tumor growth but promoted TNBC cell migration and invasion in vitro and lung metastasis in vivo. The effect of ETHE1 on TNBC cell migratory potential was independent of its enzymatic activity. Mechanistic investigations revealed that ETHE1 interacted with eIF2α and enhanced its phosphorylation by promoting the interaction between eIF2α and GCN2. Phosphorylated eIF2α in turn upregulated the expression of ATF4, a transcriptional activator of genes involved in cell migration and tumor metastasis. Notably, inhibition of eIF2α phosphorylation through ISRIB or ATF4 knockdown partially abolished the tumor-promoting effect of ETHE1 overexpression. ETHE1 has a functional and mechanistic role in TNBC metastasis and offers a new therapeutic strategy for targeting ETHE1-propelled TNBC using ISRIB.
Sujet(s)
Tumeurs du sein triple-négatives , Humains , Tumeurs du sein triple-négatives/anatomopathologie , Facteur-2 d'initiation eucaryote/métabolisme , Lignée cellulaire tumorale , Transduction du signal , Prolifération cellulaire/génétique , Mouvement cellulaire/génétique , Régulation de l'expression des gènes tumoraux , Protéines mitochondriales/métabolisme , Transporteurs nucléocytoplasmiques/métabolisme , Facteur de transcription ATF-4/génétique , Facteur de transcription ATF-4/métabolismeRÉSUMÉ
A critical step in the removal of polyubiquitinated proteins from macromolecular complexes and membranes for subsequent proteasomal degradation is the unfolding of an ubiquitin moiety by the cofactor Ufd1/Npl4 (UN) and its insertion into the Cdc48 ATPase for mechanical translocation. Here, we present a stepwise protocol for the assembly and purification of Lys48-linked ubiquitin chains that are fluorophore labeled at specific ubiquitin moieties and allow monitoring polyubiquitin engagement by the Cdc48-UN complex in a FRET-based assay. For complete details on the use and execution of this protocol, please refer to Williams et al. (2023).1.
Sujet(s)
Polyubiquitine , Protéines de Saccharomyces cerevisiae , Protéines de Saccharomyces cerevisiae/métabolisme , Protéine contenant la valosine/composition chimique , Protéine contenant la valosine/métabolisme , Transfert d'énergie par résonance de fluorescence , Transporteurs nucléocytoplasmiques/composition chimique , Transporteurs nucléocytoplasmiques/métabolisme , Ubiquitine/métabolismeRÉSUMÉ
Transmembrane p24 trafficking protein 10 (TMED10) is a conserved vesicle trafficking protein. It is dysregulated in Alzheimer disease and plays a pivotal role in the pathogenesis of Alzheimer disease. In addition to the brain, TMED10 is highly expressed in the exocrine pancreas; however, its biological functions and underlying mechanisms remain largely unknown. We studied reduced Tmed10 in zebrafish embryos by morpholino oligonucleotide knockdown and CRISPR-Cas9 mutagenesis. Tmed10-deficient embryos showed extensive loss of acinar mass and impaired acinar differentiation. TMED10 has been reported to have an inhibitory effect on γ-secretase. As one of the substrates of γ-secretase, membrane-bound ß-catenin was significantly reduced in Tmed10-deficient embryos. Increased γ-secretase activity in wild-type embryos resulted in a phenotype similar to that of tmed10 mutants. And the mutant phenotype could be rescued by treatment with the γ-secretase inhibitor, N-[N-(3, 5-difluorophenacetyl)-l-alanyl]-s-phenylglycinet-butyl ester (DAPT). In addition, the reduced membrane-bound ß-catenin was accompanied with up-regulated ß-catenin target genes in Tmed10-deficient embryos. Overexpression of ß-catenin signaling inhibitor Dickkopf-1 (DKK-1) could rescue the exocrine pancreas defects. Taken together, our study reveals that Tmed10 regulates exocrine pancreatic differentiation through γ-secretase. Reduced membrane-bound ß-catenin, accompanied with hyperactivation of ß-catenin signaling, is an important cause of exocrine pancreas defects in Tmed10-deficient embryos. Our study reaffirms the importance of appropriate ß-catenin signaling in exocrine pancreas development. These findings may provide a theoretical basis for the development of treatment strategies for TMED10-related diseases.
Sujet(s)
Maladie d'Alzheimer , Transporteurs nucléocytoplasmiques , Pancréas exocrine , Animaux , Amyloid precursor protein secretases/génétique , bêta-Caténine/génétique , Larve , Pancréas exocrine/embryologie , Pancréas exocrine/métabolisme , Danio zébré/génétique , Transporteurs nucléocytoplasmiques/métabolismeRÉSUMÉ
Cell viability largely depends on the surveillance of mRNA export and translation. Upon pre-mRNA processing and nuclear quality control, mature mRNAs are exported into the cytoplasm via Mex67-Mtr2 attachment. At the cytoplasmic site of the nuclear pore complex, the export receptor is displaced by the action of the DEAD-box RNA helicase Dbp5. Subsequent quality control of the open reading frame requires translation. Our studies suggest an involvement of Dbp5 in cytoplasmic no-go-and non-stop decay. Most importantly, we have also identified a key function for Dbp5 in translation termination, which identifies this helicase as a master regulator of mRNA expression.
Sujet(s)
Transporteurs nucléocytoplasmiques , Protéines de Saccharomyces cerevisiae , Transporteurs nucléocytoplasmiques/génétique , Transporteurs nucléocytoplasmiques/métabolisme , DEAD-box RNA helicases/génétique , DEAD-box RNA helicases/métabolisme , Transport nucléaire actif , Protéines de Saccharomyces cerevisiae/métabolisme , ARN messager/génétique , ARN messager/métabolisme , Expression des gènesRÉSUMÉ
Self-renewing somatic tissues rely on progenitors to support the continuous tissue regeneration. The gene regulatory network maintaining progenitor function remains incompletely understood. Here we show that NUP98 and RAE1 are highly expressed in epidermal progenitors, forming a separate complex in the nucleoplasm. Reduction of NUP98 or RAE1 abolishes progenitors' regenerative capacity, inhibiting proliferation and inducing premature terminal differentiation. Mechanistically, NUP98 binds on chromatin near the transcription start sites of key epigenetic regulators (such as DNMT1, UHRF1 and EZH2) and sustains their expression in progenitors. NUP98's chromatin binding sites are co-occupied by HDAC1. HDAC inhibition diminishes NUP98's chromatin binding and dysregulates NUP98 and RAE1's target gene expression. Interestingly, HDAC inhibition further induces NUP98 and RAE1 to localize interdependently to the nucleolus. These findings identified a pathway in progenitor maintenance, where HDAC activity directs the high levels of NUP98 and RAE1 to directly control key epigenetic regulators, escaping from nucleolar aggregation.
Sujet(s)
Chromatine , Transporteurs nucléocytoplasmiques , Transporteurs nucléocytoplasmiques/composition chimique , Transporteurs nucléocytoplasmiques/génétique , Transporteurs nucléocytoplasmiques/métabolisme , Chromatine/génétique , Protéines associées à la matrice nucléaire/composition chimique , Protéines associées à la matrice nucléaire/génétique , Protéines associées à la matrice nucléaire/métabolisme , Sites de fixationRÉSUMÉ
tRNAs are small noncoding RNAs that are predominantly known for their roles in protein synthesis and also participate in numerous other functions ranging from retroviral replication to apoptosis. In eukaryotic cells, all tRNAs move bidirectionally, shuttling between the nucleus and the cytoplasm. Bidirectional nuclear-cytoplasmic tRNA trafficking requires a complex set of conserved proteins. Here, we describe an in vivo biochemical methodology in Saccharomyces cerevisiae to assess the ability of proteins implicated in tRNA nuclear export to form nuclear export complexes with tRNAs. This method employs tagged putative tRNA nuclear exporter proteins and co-immunoprecipitation of tRNA-exporter complexes using antibody-conjugated magnetic beads. Because the interaction between nuclear exporters and tRNAs may be transient, this methodology employs strategies to effectively trap tRNA-protein complexes in vivo. This pull-down method can be used to verify and characterize candidate proteins and their potential interactors implicated in tRNA nuclear-cytoplasmic trafficking.
Sujet(s)
Protéines de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/génétique , Transport nucléaire actif/génétique , Transporteurs nucléocytoplasmiques/métabolisme , Protéines de Saccharomyces cerevisiae/métabolisme , ARN de transfert/génétique , Noyau de la cellule/métabolisme , Protéines nucléaires/métabolismeRÉSUMÉ
Here, we ask how developing precursors maintain the balance between cell genesis for tissue growth and establishment of adult stem cell pools, focusing on postnatal forebrain neural precursor cells (NPCs). We show that these NPCs are transcriptionally primed to differentiate and that the primed mRNAs are associated with the translational repressor 4E-T. 4E-T also broadly associates with other NPC mRNAs encoding transcriptional regulators, and these are preferentially depleted from ribosomes, consistent with repression. By contrast, a second translational regulator, Cpeb4, associates with diverse target mRNAs that are largely ribosome associated. The 4E-T-dependent mRNA association is functionally important because 4E-T knockdown or conditional knockout derepresses proneurogenic mRNA translation and perturbs maintenance versus differentiation of early postnatal NPCs in culture and in vivo. Thus, early postnatal NPCs are primed to differentiate, and 4E-T regulates the balance between cell genesis and stem cell expansion by sequestering and repressing mRNAs encoding transcriptional regulators.
Sujet(s)
Cellules souches neurales , Différenciation cellulaire/physiologie , Cellules souches neurales/métabolisme , Neurones/métabolisme , Corps de traitement , Biosynthèse des protéines , Protéines de répression/métabolisme , ARN messager/génétique , ARN messager/métabolisme , Transporteurs nucléocytoplasmiques/métabolismeRÉSUMÉ
Hydrogen sulfide (H2S) was previously revealed to inhibit osteoblastic differentiation of valvular interstitial cells (VICs), a pathological feature in calcific aortic valve disease (CAVD). This study aimed to explore the metabolic control of H2S levels in human aortic valves. Lower levels of bioavailable H2S and higher levels of interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) were detected in aortic valves of CAVD patients compared to healthy individuals, accompanied by higher expression of cystathionine γ-lyase (CSE) and same expression of cystathionine ß-synthase (CBS). Increased biogenesis of H2S by CSE was found in the aortic valves of CAVD patients which is supported by increased production of lanthionine. In accordance, healthy human aortic VICs mimic human pathology under calcifying conditions, as elevated CSE expression is associated with low levels of H2S. The expression of mitochondrial enzymes involved in H2S catabolism including sulfide quinone oxidoreductase (SQR), the key enzyme in mitochondrial H2S oxidation, persulfide dioxygenase (ETHE1), sulfite oxidase (SO) and thiosulfate sulfurtransferase (TST) were up-regulated in calcific aortic valve tissues, and a similar expression pattern was observed in response to high phosphate levels in VICs. AP39, a mitochondria-targeting H2S donor, rescued VICs from an osteoblastic phenotype switch and reduced the expression of IL-1ß and TNF-α in VICs. Both pro-inflammatory cytokines aggravated calcification and osteoblastic differentiation of VICs derived from the calcific aortic valves. In contrast, IL-1ß and TNF-α provided an early and transient inhibition of VICs calcification and osteoblastic differentiation in healthy cells and that effect was lost as H2S levels decreased. The benefit was mediated via CSE induction and H2S generation. We conclude that decreased levels of bioavailable H2S in human calcific aortic valves result from an increased H2S metabolism that facilitates the development of CAVD. CSE/H2S represent a pathway that reverses the action of calcifying stimuli.
Sujet(s)
Sténose aortique , Calcinose , Sulfure d'hydrogène , Humains , Valve aortique/métabolisme , Valve aortique/anatomopathologie , Sténose aortique/métabolisme , Sténose aortique/anatomopathologie , Sulfure d'hydrogène/métabolisme , Calcinose/métabolisme , Calcinose/anatomopathologie , Facteur de nécrose tumorale alpha/génétique , Facteur de nécrose tumorale alpha/métabolisme , Cellules cultivées , Mitochondries/métabolisme , Protéines mitochondriales/métabolisme , Transporteurs nucléocytoplasmiques/métabolismeRÉSUMÉ
The AAA+ ATPase Cdc48 utilizes the cofactor Ufd1/Npl4 to bind and thread polyubiquitinated substrates for their extraction from complexes or membranes and often for subsequent proteasomal degradation. Previous studies indicated that Cdc48 engages polyubiquitin chains through the Npl4-mediated unfolding of an initiator ubiquitin; yet, the underlying principles remain largely unknown. Using FRET-based assays, we revealed the mechanisms and kinetics of ubiquitin unfolding, insertion into the ATPase, and unfolding of the ubiquitin-attached substrate. We found that Cdc48 uses Ufd1's UT3 domain to bind a K48-linked ubiquitin on the initiator's proximal side of the chain, thereby directing the initiator toward rapid unfolding by Npl4 and engagement by Cdc48. Ubiquitins on the initiator's distal side increase substrate affinity and facilitate unfolding but impede substrate release from Cdc48-Ufd1/Npl4 in the absence of additional cofactors. Our findings explain how Cdc48-UN efficiently processes substrates with K48-linked chains of 4-6 ubiquitins, which represent most cellular polyubiquitinated proteins.
Sujet(s)
Polyubiquitine , Protéines de Saccharomyces cerevisiae , Polyubiquitine/métabolisme , Protéines de Saccharomyces cerevisiae/métabolisme , ATPases associated with diverse cellular activities/métabolisme , Protéine contenant la valosine/métabolisme , Protéines du transport vésiculaire/métabolisme , Transporteurs nucléocytoplasmiques/métabolisme , Ubiquitine/métabolisme , Ubiquitines/métabolisme , Protéines du cycle cellulaire/métabolismeRÉSUMÉ
In eukaryotic cells, various classes of RNAs are exported to the cytoplasm by class-specific factors. Accumulating evidence has shown that export factors affect the fate of RNA, demonstrating the importance of proper RNA classification upon export. We previously reported that RNA polymerase II transcripts were classified after synthesis depending on their length, and identified heterogeneous nuclear ribonucleoprotein (hnRNP) C as the key classification factor. HnRNP C inhibits the recruitment of PHAX, an adapter protein for spliceosomal U snRNA export, to long transcripts, navigating these RNAs to the mRNA export pathway. However, the mechanisms by which hnRNP C inhibits PHAX recruitment to mRNA remain unknown. We showed that the cap-binding complex, a bridging factor between m7G-capped RNA and PHAX, directly interacted with hnRNP C on mRNA. Additionally, we revealed that the tetramer-forming activity of hnRNP C and its strong RNA-binding activity were crucial for the inhibition of PHAX binding to longer RNAs. These results suggest that mRNA is wrapped around the hnRNP C tetramer without a gap from the cap, thereby impeding the recruitment of PHAX. The results obtained on the mode of length-specific RNA classification by the hnRNP C tetramer will provide mechanistic insights into hnRNP C-mediated RNA biogenesis.
Sujet(s)
Ribonucléoprotéine nucléaire hétérogène du groupe C , RNA polymerase II , Ribonucléoprotéine nucléaire hétérogène du groupe C/métabolisme , Transporteurs nucléocytoplasmiques/métabolisme , RNA polymerase II/métabolisme , ARN messager/métabolisme , Petit ARN nucléaire/génétique , Cellules eucaryotes/métabolismeRÉSUMÉ
The changes occurring in mRNA organization during nucleo-cytoplasmic transport and export, are not well understood. Moreover, directionality of mRNA passage through the nuclear pore complex (NPC) has not been examined within individual NPCs. Here we find that an mRNP is compact during nucleoplasmic travels compared to a more open structure after transcription and at the nuclear periphery. Compaction levels of nuclear transcripts can be modulated by varying levels of SR proteins and by changing genome organization. Nuclear mRNPs are mostly rod-shaped with distant 5'/3'-ends, although for some, the ends are in proximity. The latter is more abundant in the cytoplasm and can be modified by translation inhibition. mRNAs and lncRNAs exiting the NPC exhibit predominant 5'-first export. In some cases, several adjacent NPCs are engaged in export of the same mRNA suggesting 'gene gating'. Altogether, we show that the mRNP is a flexible structure during travels, with 5'-directionality during export.