Anaplastic large cell lymphoma (ALCL), an aggressive CD30-positive T-cell lymphoma, comprises systemic anaplastic lymphoma kinase (ALK)-positive, and ALK-negative, primary cutaneous and breast implant-associated ALCL. Prognosis of some ALCL subgroups is still unsatisfactory, and already in second line effective treatment options are lacking. To identify genes defining ALCL cell state and dependencies, we here characterize super-enhancer regions by genome-wide H3K27ac ChIP-seq. In addition to known ALCL key regulators, the AP-1-member BATF3 and IL-2 receptor (IL2R)-components are among the top hits. Specific and high-level IL2R expression in ALCL correlates with BATF3 expression. Confirming a regulatory link, IL-2R-expression decreases following BATF3 knockout, and BATF3 is recruited to IL2R regulatory regions. Functionally, IL-2, IL-15 and Neo-2/15, a hyper-stable IL-2/IL-15 mimic, accelerate ALCL growth and activate STAT1, STAT5 and ERK1/2. In line, strong IL-2Rα-expression in ALCL patients is linked to more aggressive clinical presentation. Finally, an IL-2Rα-targeting antibody-drug conjugate efficiently kills ALCL cells in vitro and in vivo. Our results highlight the importance of the BATF3/IL-2R-module for ALCL biology and identify IL-2Rα-targeting as a promising treatment strategy for ALCL.
- MeSH
- anaplastický velkobuněčný lymfom farmakoterapie genetika metabolismus patologie MeSH
- antigen Ki-1 genetika metabolismus MeSH
- imunokonjugáty farmakologie MeSH
- interleukin-15 farmakologie MeSH
- interleukin-2 farmakologie MeSH
- lidé MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- proliferace buněk účinky léků MeSH
- receptor interleukinu-2 - alfa-podjednotka genetika imunologie metabolismus MeSH
- receptory interleukinu-2 genetika imunologie metabolismus MeSH
- regulace genové exprese u nádorů MeSH
- regulační oblasti nukleových kyselin MeSH
- represorové proteiny genetika metabolismus MeSH
- signální transdukce účinky léků MeSH
- transkripční faktory bZIP genetika metabolismus MeSH
- viabilita buněk účinky léků MeSH
- xenogenní modely - testy antitumorózní aktivity MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Preeclampsia (PE) is a major cause of the pregnancy morbidity and mortality over the world. Disorganized placentation caused by trophoblast cell abnormity is one of main risk factors to induce PE. MiR-133a-3p has been shown to contain regulatory effects on oxidative stress in the cardiomyocytes. But the effects of miR-133a-3p on oxidative stress-induced apoptosis in the trophoblast cells remain unknown. In this study, trophoblast HTR-8/SVneo cells were transfected with miR-133a-3p mimics and inhibitor. H2O2 (250 microM) treatment of cells was adopted to induce oxidative stress. A series of typical molecular and cellular experiments was subsequently performed in order to investigate this issue. It was found that miR-133a-3p overexpression attenuated the oxidative stress induced by H2O2 through reduced ROS and MDA levels and enhanced antioxidase activities in the trophoblast cells. Overexpressed miR-133a-3p was shown to relieve the oxidative stress-induced apoptosis of HTR-8/SVneo cells. At molecular levels, a direct binding effect of miR-133a-3p on BACH1 was verified. Moreover, miR-133a-3p overexpression also enhanced BACH1 downstream Nrf2/HO-1 signaling to activate antioxidant genes. It is collectively demonstrated that miR-133a-3p can relieve the oxidative stress-induced apoptosis in the trophoblast cells through the BACH1/Nrf2/HO-1 signaling pathway via targeting BACH1 directly. This regulatory mechanism of miR-133a-3p in the trophoblast cells under oxidative stress may give a new perspective for oxidative stress-induced trophoblast cell abnormality and be useful to study more pathological mechanisms of PE.
- MeSH
- apoptóza účinky léků MeSH
- buněčné linie MeSH
- faktor 2 související s NF-E2 metabolismus MeSH
- hemoxygenasa-1 metabolismus MeSH
- lidé MeSH
- mikro RNA aplikace a dávkování genetika MeSH
- oxidační stres účinky léků MeSH
- preeklampsie metabolismus patologie prevence a kontrola MeSH
- signální transdukce MeSH
- těhotenství MeSH
- transkripční faktory bZIP metabolismus MeSH
- trofoblasty účinky léků metabolismus patologie MeSH
- Check Tag
- lidé MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
Translational control targeting the initiation phase is central to the regulation of gene expression. Understanding all of its aspects requires substantial technological advancements. Here we modified yeast translation complex profile sequencing (TCP-seq), related to ribosome profiling, and adapted it for mammalian cells. Human TCP-seq, capable of capturing footprints of 40S subunits (40Ss) in addition to 80S ribosomes (80Ss), revealed that mammalian and yeast 40Ss distribute similarly across 5'TRs, indicating considerable evolutionary conservation. We further developed yeast and human selective TCP-seq (Sel-TCP-seq), enabling selection of 40Ss and 80Ss associated with immuno-targeted factors. Sel-TCP-seq demonstrated that eIF2 and eIF3 travel along 5' UTRs with scanning 40Ss to successively dissociate upon AUG recognition; notably, a proportion of eIF3 lingers on during the initial elongation cycles. Highlighting Sel-TCP-seq versatility, we also identified four initiating 48S conformational intermediates, provided novel insights into ATF4 and GCN4 mRNA translational control, and demonstrated co-translational assembly of initiation factor complexes.
- MeSH
- 5' nepřekládaná oblast MeSH
- eukaryotický iniciační faktor 2 genetika metabolismus MeSH
- eukaryotický iniciační faktor 3 genetika metabolismus MeSH
- HEK293 buňky MeSH
- iniciační faktory genetika metabolismus MeSH
- kodon iniciační MeSH
- lidé MeSH
- malé podjednotky ribozomu eukaryotické genetika metabolismus MeSH
- multiproteinové komplexy genetika metabolismus MeSH
- proteosyntéza * MeSH
- ribozomy genetika metabolismus MeSH
- Saccharomyces cerevisiae - proteiny genetika metabolismus MeSH
- Saccharomyces cerevisiae genetika MeSH
- transkripční faktor ATF4 genetika metabolismus MeSH
- transkripční faktory bZIP genetika metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
KEY MESSAGE : bZIP TF network in pollen. Transcriptional control of gene expression represents an important mechanism guiding organisms through developmental processes and providing plasticity towards environmental stimuli. Because of their sessile nature, plants require effective gene regulation for rapid response to variation in environmental and developmental conditions. Transcription factors (TFs) provide such control ensuring correct gene expression in spatial and temporal manner. Our work reports the interaction network of six bZIP TFs expressed in Arabidopsis thaliana pollen and highlights the potential functional role for AtbZIP18 in pollen. AtbZIP18 was shown to interact with three other pollen-expressed bZIP TFs-AtbZIP34, AtbZIP52, and AtbZIP61 in yeast two-hybrid assays. AtbZIP18 transcripts are highly expressed in pollen, and at the subcellular level, an AtbZIP18-GFP fusion protein was located in the nucleus and cytoplasm/ER. To address the role of AtbZIP18 in the male gametophyte, we performed phenotypic analysis of a T-DNA knockout allele, which showed slightly reduced transmission through the male gametophyte. Some of the phenotype defects in atbzip18 pollen, although observed at low penetrance, were similar to those seen at higher frequency in the T-DNA knockout of the interacting partner, AtbZIP34. To gain deeper insight into the regulatory role of AtbZIP18, we analysed atbzip18/- pollen microarray data. Our results point towards a potential repressive role for AtbZIP18 and its functional redundancy with AtbZIP34 in pollen.
- MeSH
- Arabidopsis cytologie metabolismus ultrastruktura MeSH
- dimerizace MeSH
- DNA rostlinná MeSH
- inzerční mutageneze MeSH
- proteiny huseníčku metabolismus MeSH
- pyl genetika růst a vývoj metabolismus ultrastruktura MeSH
- regulace genové exprese u rostlin MeSH
- rekombinantní fúzní proteiny metabolismus MeSH
- trans-aktivátory metabolismus MeSH
- transkripční faktory bZIP metabolismus MeSH
- vazba proteinů MeSH
- Publikační typ
- časopisecké články MeSH
Translational control in eukaryotes is exerted by many means, one of which involves a ribosome translating multiple cistrons per mRNA as in bacteria. It is called reinitiation (REI) and occurs on mRNAs where the main ORF is preceded by a short upstream uORF(s). Some uORFs support efficient REI on downstream cistrons, whereas some others do not. The mRNA of yeast transcriptional activator GCN4 contains four uORFs of both types that together compose an intriguing regulatory mechanism of its expression responding to nutrients' availability and various stresses. Here we subjected all GCN4 uORFs to a comprehensive analysis to identify all REI-promoting and inhibiting cis-determinants that contribute either autonomously or in synergy to the overall efficiency of REI on GCN4. We found that the 3' sequences of uORFs 1-3 contain a conserved AU1-2A/UUAU2 motif that promotes REI in position-specific, autonomous fashion such as the REI-promoting elements occurring in 5' sequences of uORF1 and uORF2. We also identified autonomous and transferable REI-inhibiting elements in the 3' sequences of uORF2 and uORF3, immediately following their AU-rich motif. Furthermore, we analyzed contributions of coding triplets and terminating stop codon tetranucleotides of GCN4 uORFs showing a negative correlation between the efficiency of reinitiation and efficiency of translation termination. Together we provide a complex overview of all cis-determinants of REI with their effects set in the context of the overall GCN4 translational control.
- MeSH
- iniciace translace peptidového řetězce MeSH
- messenger RNA genetika metabolismus MeSH
- otevřené čtecí rámce MeSH
- regulace genové exprese u hub MeSH
- Saccharomyces cerevisiae - proteiny genetika metabolismus MeSH
- Saccharomyces cerevisiae genetika metabolismus MeSH
- sekvence nukleotidů MeSH
- sekvenční analýza RNA MeSH
- transkripční faktory bZIP genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Despite the completion of the Arabidopsis genome sequence, for only a relatively low percentage of the encoded proteins experimental evidence concerning their function is available. Plant proteins that harbour a single PLAT (Polycystin, Lipoxygenase, Alpha-toxin and Triacylglycerol lipase) domain and belong to the PLAT-plant-stress protein family are ubiquitously present in monocot and dicots. However, the function of PLAT-plant-stress proteins is still poorly understood. Therefore, we have assessed the function of the uncharacterised Arabidopsis PLAT-plant-stress family members through a combination of functional genetic and physiological approaches. PLAT1 overexpression conferred increased abiotic stress tolerance, including cold, drought and salt stress, while loss-of-function resulted in opposite effects on abiotic stress tolerance. Strikingly, PLAT1 promoted growth under non-stressed conditions. Abiotic stress treatments induced PLAT1 expression and caused expansion of its expression domain. The ABF/ABRE transcription factors, which are positive mediators of abscisic acid signalling, activate PLAT1 promoter activity in transactivation assays and directly bind to the ABRE elements located in this promoter in electrophoretic mobility shift assays. This suggests that PLAT1 represents a novel downstream target of the abscisic acid signalling pathway. Thus, we showed that PLAT1 critically functions as positive regulator of abiotic stress tolerance, but also is involved in regulating plant growth, and thereby assigned a function to this previously uncharacterised PLAT domain protein. The functional data obtained for PLAT1 support that PLAT-plant-stress proteins in general could be promising targets for improving abiotic stress tolerance without yield penalty.
- MeSH
- aktivace transkripce MeSH
- Arabidopsis klasifikace růst a vývoj metabolismus MeSH
- fylogeneze MeSH
- fyziologický stres * MeSH
- kyselina abscisová farmakologie MeSH
- nízká teplota MeSH
- období sucha MeSH
- promotorové oblasti (genetika) MeSH
- proteiny huseníčku genetika metabolismus MeSH
- regulace genové exprese u rostlin účinky léků MeSH
- retardační test MeSH
- signální transdukce účinky léků MeSH
- soli chemie farmakologie MeSH
- stres endoplazmatického retikula účinky léků MeSH
- transkripční faktory bZIP genetika metabolismus MeSH
- tunikamycin toxicita MeSH
- vazba proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
One of the extensively studied mechanisms of gene-specific translational regulation is reinitiation. It takes place on messenger RNAs (mRNAs) where main ORF is preceded by upstream ORF (uORF). Even though uORFs generally down-regulate main ORF expression, specific uORFs exist that allow high level of downstream ORF expression. The key is their ability to retain 40S subunits on mRNA upon termination of their translation to resume scanning for the next AUG. Here, we took advantage of the exemplary model system of reinitiation, the mRNA of yeast transcriptional activator GCN4 containing four short uORFs, and show that contrary to previous reports, not only the first but the first two of its uORFs allow efficient reinitiation. Strikingly, we demonstrate that they utilize a similar molecular mechanism relying on several cis-acting 5' reinitiation-promoting elements, one of which they share, and the interaction with the a/TIF32 subunit of translation initiation factor eIF3. Since a similar mechanism operates also on YAP1 uORF, our findings strongly suggest that basic principles of reinitiation are conserved. Furthermore, presence of two consecutive reinitiation-permissive uORFs followed by two reinitiation-non-permissive uORFs suggests that tightness of GCN4 translational control is ensured by a fail-safe mechanism that effectively prevents or triggers GCN4 expression under nutrient replete or deplete conditions, respectively.
- MeSH
- 5' nepřekládaná oblast MeSH
- eukaryotický iniciační faktor 3 metabolismus MeSH
- fungální RNA genetika metabolismus MeSH
- iniciace translace peptidového řetězce * MeSH
- messenger RNA genetika metabolismus MeSH
- molekulární sekvence - údaje MeSH
- otevřené čtecí rámce MeSH
- regulace genové exprese u hub MeSH
- Saccharomyces cerevisiae - proteiny genetika metabolismus MeSH
- Saccharomyces cerevisiae genetika metabolismus MeSH
- sekvence nukleotidů MeSH
- transkripční faktory bZIP genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Reinitiation is a gene-specific translational control mechanism characterized by the ability of some short upstream uORFs to retain post-termination 40S subunits on mRNA. Its efficiency depends on surrounding cis-acting sequences, uORF elongation rates, various initiation factors, and the intercistronic distance. To unravel effects of cis-acting sequences, we investigated previously unconsidered structural properties of one such a cis-enhancer in the mRNA leader of GCN4 using yeast genetics and biochemistry. This leader contains four uORFs but only uORF1, flanked by two transferrable 5' and 3' cis-acting sequences, and allows efficient reinitiation. Recently we showed that the 5' cis-acting sequences stimulate reinitiation by interacting with the N-terminal domain (NTD) of the eIF3a/TIF32 subunit of the initiation factor eIF3 to stabilize post-termination 40S subunits on uORF1 to resume scanning downstream. Here we identify four discernible reinitiation-promoting elements (RPEs) within the 5' sequences making up the 5' enhancer. Genetic epistasis experiments revealed that two of these RPEs operate in the eIF3a/TIF32-dependent manner. Likewise, two separate regions in the eIF3a/TIF32-NTD were identified that stimulate reinitiation in concert with the 5' enhancer. Computational modeling supported by experimental data suggests that, in order to act, the 5' enhancer must progressively fold into a specific secondary structure while the ribosome scans through it prior uORF1 translation. Finally, we demonstrate that the 5' enhancer's stimulatory activity is strictly dependent on and thus follows the 3' enhancer's activity. These findings allow us to propose for the first time a model of events required for efficient post-termination resumption of scanning. Strikingly, structurally similar RPE was predicted and identified also in the 5' leader of reinitiation-permissive uORF of yeast YAP1. The fact that it likewise operates in the eIF3a/TIF32-dependent manner strongly suggests that at least in yeasts the underlying mechanism of reinitiation on short uORFs is conserved.
- MeSH
- 5' nepřekládaná oblast MeSH
- 5' přiléhající oblast DNA MeSH
- DNA vazebné proteiny genetika metabolismus MeSH
- eukaryotický iniciační faktor 3 genetika metabolismus MeSH
- malé podjednotky ribozomu eukaryotické genetika metabolismus MeSH
- messenger RNA genetika metabolismus MeSH
- otevřené čtecí rámce genetika MeSH
- regulační oblasti nukleových kyselin MeSH
- ribozomální proteiny genetika metabolismus MeSH
- ribozomy genetika metabolismus MeSH
- Saccharomyces cerevisiae - proteiny genetika metabolismus MeSH
- Saccharomyces cerevisiae genetika metabolismus MeSH
- sekvence nukleotidů MeSH
- transkripční faktory bZIP genetika metabolismus MeSH
- transkripční faktory genetika metabolismus MeSH
- zesilovače transkripce MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Recent reports have begun unraveling the details of various roles of individual eukaryotic translation initiation factor 3 (eIF3) subunits in translation initiation. Here we describe functional characterization of two essential Saccharomyces cerevisiae eIF3 subunits, g/Tif35 and i/Tif34, previously suggested to be dispensable for formation of the 48S preinitiation complexes (PICs) in vitro. A triple-Ala substitution of conserved residues in the RRM of g/Tif35 (g/tif35-KLF) or a single-point mutation in the WD40 repeat 6 of i/Tif34 (i/tif34-Q258R) produces severe growth defects and decreases the rate of translation initiation in vivo without affecting the integrity of eIF3 and formation of the 43S PICs in vivo. Both mutations also diminish induction of GCN4 expression, which occurs upon starvation via reinitiation. Whereas g/tif35-KLF impedes resumption of scanning for downstream reinitiation by 40S ribosomes terminating at upstream open reading frame 1 (uORF1) in the GCN4 mRNA leader, i/tif34-Q258R prevents full GCN4 derepression by impairing the rate of scanning of posttermination 40S ribosomes moving downstream from uORF1. In addition, g/tif35-KLF reduces processivity of scanning through stable secondary structures, and g/Tif35 specifically interacts with Rps3 and Rps20 located near the ribosomal mRNA entry channel. Together these results implicate g/Tif35 and i/Tif34 in stimulation of linear scanning and, specifically in the case of g/Tif35, also in proper regulation of the GCN4 reinitiation mechanism.
- MeSH
- aminokyselinové motivy genetika MeSH
- eukaryotický iniciační faktor 3 chemie genetika metabolismus MeSH
- fungální RNA metabolismus MeSH
- messenger RNA genetika metabolismus MeSH
- molekulární modely MeSH
- molekulární sekvence - údaje MeSH
- mutace MeSH
- podjednotky proteinů chemie genetika metabolismus MeSH
- proteosyntéza MeSH
- ribozomální proteiny chemie genetika metabolismus MeSH
- ribozomy genetika metabolismus MeSH
- Saccharomyces cerevisiae - proteiny chemie genetika metabolismus MeSH
- Saccharomyces cerevisiae genetika metabolismus MeSH
- sekundární struktura proteinů MeSH
- sekvence aminokyselin MeSH
- sekvenční homologie aminokyselin MeSH
- substituce aminokyselin MeSH
- terciární struktura proteinů MeSH
- transkripční faktory bZIP genetika metabolismus MeSH
- vazba proteinů MeSH
- western blotting MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH