scholarly journals Molecular mechanism of translational stalling by inhibitory codon combinations and poly(A) tracts

2019 ◽  
Author(s):  
Petr Tesina ◽  
Laura N. Lessen ◽  
Robert Buschauer ◽  
Jingdong Cheng ◽  
Colin Chih-Chien Wu ◽  
...  
Keyword(s):  
Molecular Mechanism ◽  
Molecular Mechanisms ◽  
Ribosome Profiling ◽  
Translation System ◽  
Structural Explanation ◽  
Ribosome Stalling ◽  
A Site ◽  
Codon Pairs ◽  
Protein Output

Inhibitory codon pairs and poly(A) tracts within the translated mRNA cause ribosome stalling and reduce protein output. The molecular mechanisms that drive these stalling events, however, are still unknown. Here, we use a combination ofin vitrobiochemistry, ribosome profiling, and cryo-EM to define molecular mechanisms that lead to these ribosome stalls. First, we use anin vitroreconstituted yeast translation system to demonstrate that inhibitory codon pairs slow elongation rates which are partially rescued by increased tRNA concentration or by an artificial tRNA not dependent on wobble base pairing. Ribosome profiling data extend these observations by revealing that paused ribosomes with empty A sites are enriched on these sequences. Cryo-EM structures of stalled ribosomes provide a structural explanation for the observed effects by showing decoding-incompatible conformations of mRNA in the A sites of all studied stall-inducing sequences. Interestingly, in the case of poly(A) tracts, the inhibitory conformation of the mRNA in the A site involves a nucleotide stacking array. Together, these data demonstrate novel mRNA-induced mechanisms of translational stalling in eukaryotic ribosomes.

scholarly journals In Vitro Reconstitution of Yeast Translation System Capable of Synthesizing Long Polypeptide and Recapitulating Programmed Ribosome Stalling

2021 ◽  
Vol 4 (3) ◽  
pp. 45
Author(s):  
Riku Nagai ◽  
Yichen Xu ◽  
Chang Liu ◽  
Ayaka Shimabukuro ◽  
Nono Takeuchi-Tomita
Keyword(s):  
Unnatural Amino Acids ◽  
Molecular Mechanisms ◽  
In Vitro Translation ◽  
Critical Parameters ◽  
Translation System ◽  
Reaction Conditions ◽  
Translation Elongation ◽  
Functional Studies ◽  
Ribosome Stalling

The rates of translation elongation or termination in eukaryotes are modulated through cooperative molecular interactions involving mRNA, the ribosome, aminoacyl- and nascent polypeptidyl-tRNAs, and translation factors. To investigate the molecular mechanisms underlying these processes, we developed an in vitro translation system from yeast, reconstituted with purified translation elongation and termination factors, utilizing CrPV IGR IRES-containing mRNA, which functions in the absence of initiation factors. The system is capable of synthesizing not only short oligopeptides but also long reporter proteins such as nanoluciferase. By setting appropriate translation reaction conditions, such as the Mg2+/polyamine concentration, the arrest of translation elongation by known ribosome-stalling sequences (e.g., polyproline and CGA codon repeats) is properly recapitulated in this system. We describe protocols for the preparation of the system components, manipulation of the system, and detection of the translation products. We also mention critical parameters for setting up the translation reaction conditions. This reconstituted translation system not only facilitates biochemical analyses of translation but is also useful for various applications, such as structural and functional studies with the aim of designing drugs that act on eukaryotic ribosomes, and the development of systems for producing novel functional proteins by incorporating unnatural amino acids by eukaryotic ribosomes.

scholarly journals NLRP3 phosphorylation in its LRR domain critically regulates inflammasome assembly

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Tingting Niu ◽  
Charlotte De Rosny ◽  
Séverine Chautard ◽  
Amaury Rey ◽  
Danish Patoli ◽  
...  
Keyword(s):  
Molecular Mechanism ◽  
Inflammatory Cytokines ◽  
Molecular Mechanisms ◽  
Caspase 1 ◽  
Interaction Surface ◽  
Lrr Domain ◽  
Inflammasome Activity

AbstractNLRP3 controls the secretion of inflammatory cytokines IL-1β/18 and pyroptosis by assembling the inflammasome. Upon coordinated priming and activation stimuli, NLRP3 recruits NEK7 within hetero-oligomers that nucleate ASC and caspase-1 filaments, but the apical molecular mechanisms underlying inflammasome assembly remain elusive. Here we show that NEK7 recruitment to NLRP3 is controlled by the phosphorylation status of NLRP3 S803 located within the interaction surface, in which NLRP3 S803 is phosphorylated upon priming and later dephosphorylated upon activation. Phosphomimetic substitutions of S803 abolish NEK7 recruitment and inflammasome activity in macrophages in vitro and in vivo. In addition, NLRP3-NEK7 binding is also essential for NLRP3 deubiquitination by BRCC3 and subsequently inflammasome assembly, with NLRP3 phosphomimetic mutants showing enhanced ubiquitination and degradation than wildtype NLRP3. Finally, we identify CSNK1A1 as the kinase targeting NLRP3 S803. Our findings thus reveal NLRP3 S803 phosphorylation status as a druggable apical molecular mechanism controlling inflammasome assembly.

Inactivation of p34cdc2 kinase by the accumulation of its phosphorylated forms in porcine oocytes matured and aged in vitro

Zygote ◽  
1999 ◽  
Vol 7 (2) ◽  
pp. 173-179 ◽  
Author(s):  
Kazuhiro Kikuchi ◽  
Kunihiko Naito ◽  
Junko Noguchi ◽  
Arata Shimada ◽  
Hiroyuki Kaneko ◽  
...  
Keyword(s):  
Molecular Mechanism ◽  
Molecular Mechanisms ◽  
Gradual Increase ◽  
Phosphorylation Site ◽  
Catalytic Subunit ◽  
Cyclin B ◽  
Oocyte Activation ◽  
Maturation Promoting Factor

Culturing of matured porcine oocytes in vitro results in the enhancement of their cytoplasmic ability for oocyte activation (so-called ageing), although they are arrested at metaphase II. The enhanced ability for oocyte activation is related to decreased activity of the maturation promoting factor (MPF). In the present study we clarified the molecular mechanism of MPF inactivation during ageing, especially the changes in the phosphorylation status of p34cdc2, a catalytic subunit of MPF, compared with that in fertilised oocytes. The MPF activity decreased gradually when maturation culture was prolonged from 36 to 72 h, confirming the decreasing MPF activity in aged oocytes. The activity of 48 h matured oocytes also decreased after in vitro fertilisation. Immunoblotting of p34cdc2 with anti-PSTAIRE antibody revealed that the culturing of matured oocytes induces a gradual increase in pre-MPF, which is a p34cdc2 and cyclin B complex inactivated by phosphorylation at the inhibitory phosphorylation site of p34cdc2. In contrast, pre-MPF decreased after fertilisation, indicating the degradation of cyclin B. These results suggest that the molecular mechanisms of inactivation of MPF are different between oocyte activation and ageing, and that the mechanism during ageing might be based on the inhibitory phosphorylation of p34cdc2, whereas that of oocyte activation is based on the degradation of cyclin B.

scholarly journals Induction of ferroptosis in human nasopharyngeal cancer cells by cucurbitacin B: molecular mechanism and therapeutic potential

2021 ◽  
Vol 12 (3) ◽  
Author(s):  
Shuai Huang ◽  
Bihui Cao ◽  
Jinling Zhang ◽  
Yunfei Feng ◽  
Lu Wang ◽  
...  
Keyword(s):  
Molecular Mechanism ◽  
Molecular Mechanisms ◽  
Therapeutic Potential ◽  
Tumour Progression ◽  
Biological Activities ◽  
Nasopharyngeal Cancer ◽  
Migration And Invasion ◽  
Cucurbitacin B

AbstractCucurbitacin B (CuB) is a widely available triterpenoid molecule that exhibits various biological activities. Previous studies on the anti-tumour mechanism of CuB have mostly focused on cell apoptosis, and research on the ferroptosis-inducing effect has rarely been reported. Herein, we first discovered the excellent cytotoxicity of CuB towards human nasopharyngeal carcinoma cells and elucidated its potential ferroptosis-inducing mechanisms. Morphology alterations of mitochondrial ultrastructure, as observed via transmission electron microscopy, showed that CuB-treated cells undergo ferroptosis. CuB caused intracellular accumulation of iron ions and depletion of glutathione. Detailed molecular mechanism investigation confirmed that CuB both induced widespread lipid peroxidation and downregulated the expression of GPX4, ultimately initiating a multipronged mechanism of ferroptosis. Furthermore, CuB exhibited anti-tumour effects in vitro by inhibiting cellular microtubule polymerization, arresting cell cycle and suppressing migration and invasion. Finally, CuB significantly inhibited tumour progression without causing obvious side effects in vivo. Altogether, our study highlighted the therapeutic potential of CuB as a ferroptosis-inducing agent for nasopharyngeal cancer, and it provided valuable insights for developing effective anti-tumour agents with novel molecular mechanisms derived from natural products.

scholarly journals Molecular mechanism of modulating miR482b level in tomato with botrytis cinerea infection

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Fangli Wu ◽  
Jinfeng Xu ◽  
Tiantian Gao ◽  
Diao Huang ◽  
Weibo Jin
Keyword(s):  
Botrytis Cinerea ◽  
Molecular Mechanism ◽  
Molecular Mechanisms ◽  
Expression Patterns ◽  
Biotic And Abiotic Stress ◽  
Transcription Level ◽  
Expression Levels ◽  
Resistance To Stress ◽  
Rapid Amplification

Abstract Background Plant miRNAs are involved in the response to biotic and abiotic stresses by altering their expression levels, and they play an important role in the regulation of plant resistance to stress. However, the molecular mechanism that regulates the expression levels of miRNAs in plants with biotic and abiotic stress still needs to be explored. Previously, we found that the expression of the miR482 family was changed in tomato infected by Botrytis cinerea. In this study, we investigated and uncovered the mechanism underlying the response of miR482 to B. cinerea infection in tomato. Results First, RT-qPCR was employed to detect the expression patterns of miR482b in tomato infected by B. cinerea, and results showed that miR482b primary transcripts (pri-miR482b) were up-regulated in B. cinerea-infected leaves, but the mature miR482b was down-regulated. Subsequently, we used rapid amplification cDNA end method to amplify the full-length of pri-miR482b. Result showed that the pri-miR482b had two isoforms, with the longer one (consisting 300 bp) having an extra fragment of 53 bp in the 3’-end compared with the shorter one. In vitro Dicer assay indicated that the longer isoform pri-miR482b-x1 had higher efficiency in the post-transcriptional splicing of miRNA than the shorter isoform pri-miR482b-x2. In addition, the transcription level of mature miR482b was much higher in transgenic Arabidopsis overexpressing pri-miR482b-x1 than that in OE pri-miR482b-x2 Arabidopsis. These results confirmed that this extra 53 bp in pri-miR482b-x1 might play a key role in the miR482b biogenesis of post-transcription processing. Conclusions Extra 53 bp in pri-miR482b-x1 enhanced miR482b biogenesis, which elevated the transcription level of miR482b. This study clarified the response of miR482 to B. cinerea infection in tomato, thereby helping us further understand the molecular mechanisms that regulate the expression levels of other miRNAs.

scholarly journals Human NMD ensues independently of stable ribosome stalling

2019 ◽  
Author(s):  
Evangelos D. Karousis ◽  
Lukas-Adrian Gurzeler ◽  
Giuditta Annibaldis ◽  
René Dreos ◽  
Oliver Mühlemann
Keyword(s):  
Rna Degradation ◽  
Degradation Pathway ◽  
Human Cells ◽  
Ribosome Profiling ◽  
Termination Codon ◽  
Ribosome Stalling ◽  
Nonsense Mediated Mrna Decay ◽  
Similar Density

AbstractNonsense-mediated mRNA decay (NMD) is a translation-dependent RNA degradation pathway that is important for the elimination of faulty and the regulation of normal mRNAs. The molecular details of the early steps in NMD are not fully understood but previous work suggests that NMD activation occurs as a consequence of ribosome stalling at the termination codon (TC). To test this hypothesis, we established an in vitro translation-coupled toeprinting assay based on lysates from human cells that allows monitoring of ribosome occupancy at the TC of reporter mRNAs. In contrast to the prevailing NMD model, our in vitro system revealed similar ribosomal occupancy at the stop codons of NMD-sensitive and NMD-insensitive reporter mRNAs. Moreover, ribosome profiling revealed a similar density of ribosomes at the TC of endogenous NMD-sensitive and NMD-insensitive mRNAs in vivo. Together, these data show that NMD activation is not accompanied by stable stalling of ribosomes at TCs.

scholarly journals Further in Vitro Exploration Fails to Support the Allosteric Three-site Model

2012 ◽  
Vol 287 (15) ◽  
pp. 11642-11648 ◽  
Author(s):  
Alexandros D. Petropoulos ◽  
Rachel Green
Keyword(s):  
Escherichia Coli ◽  
Translation System ◽  
Large Set ◽  
Ongoing Debate ◽  
Allosteric Coupling ◽  
Site Model ◽  
A Site ◽  
Cognate Trna

Ongoing debate in the ribosome field has focused on the role of bound E-site tRNA and the Shine-Dalgarno-anti-Shine-Dalgarno (SD-aSD) interaction on A-site tRNA interactions and the fidelity of tRNA selection. Here we use an in vitro reconstituted Escherichia coli translation system to explore the reported effects of E-site-bound tRNA and SD-aSD interactions on tRNA selection events and find no evidence for allosteric coupling. A large set of experiments exploring the role of the E-site tRNA in miscoding failed to recapitulate the observations of earlier studies (Di Giacco, V., Márquez, V., Qin, Y., Pech, M., Triana-Alonso, F. J., Wilson, D. N., and Nierhaus, K. H. (2008) Proc. Natl. Acad. Sci. U.S.A. 105, 10715–10720 and Geigenmüller, U., and Nierhaus, K. H. (1990) EMBO J. 9, 4527–4533); the frequency of miscoding was unaffected by the presence of E-site-bound cognate tRNA. Moreover, our data provide clear evidence that the reported effects of the SD-aSD interaction on fidelity can be attributed to the binding of ribosomes to an unanticipated site on the mRNA (in the absence of the SD sequence) that provides a cognate pairing codon leading naturally to incorporation of the purported “noncognate” amino acid.

scholarly journals Loss of N1-methylation of G37 in tRNA induces ribosome stalling and reprograms gene expression

2021 ◽  
Author(s):  
Isao Masuda ◽  
Jae-Yeon Hwang ◽  
Thomas Christian ◽  
Sunita Maharjan ◽  
Fuad Mohammad ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Synthesis ◽  
Stringent Response ◽  
Peptide Bond ◽  
Ribosome Profiling ◽  
Peptide Bond Formation ◽  
Trna Aminoacylation ◽  
Ribosome Stalling ◽  
A Genome ◽  
A Site

N1-methylation of G37 is required for a subset of tRNAs to maintain the translational reading frame. While loss of m1G37 increases ribosomal +1 frameshifting, whether it incurs additional translational defects is unknown. Here we address this question by applying ribosome profiling to gain a genome-wide view of the effects of m1G37 deficiency on protein synthesis. Using E. coli as a model, we show that m1G37 deficiency induces ribosome stalling at codons that are normally translated by m1G37-containing tRNAs. Stalling occurs during decoding of affected codons at the ribosomal A site, indicating a distinct mechanism than that of +1 frameshifting, which occurs after the A site. Enzyme and cell-based assays show that m1G37 deficiency reduces tRNA aminoacylation and in some cases peptide-bond formation. We observe changes of gene expression in m1G37 deficiency similar to those in the starvation-induced stringent response, consistent with the notion that loss of tRNA aminoacylation activates the response. This work demonstrates a previously unrecognized function of m1G37 that emphasizes its role throughout the entire elongation cycle of protein synthesis, providing new insight into its essentiality for bacterial growth and survival.

scholarly journals PolG Inhibits Glycolysis by Suppressing PKM2 Phosphorylation Resulting Reduced Gastric Cancer Proliferation

2020 ◽  
Author(s):  
Mengzhu Lv ◽  
Simeng Zhang ◽  
Yuqing Dong ◽  
Liu Cao ◽  
Shu Guo
Keyword(s):  
Gastric Cancer ◽  
Molecular Mechanism ◽  
Molecular Mechanisms ◽  
Tumor Therapy ◽  
Tumor Formation ◽  
Cell Counting ◽  
Mechanism Analysis ◽  
Gastric Cancer Cells ◽  
The Impact

Abstract Background: Gastric cancer (GC) is the fifth most frequently diagnosed cancer and the third leading cause of cancer death. There is a critical need for the development of novel therapies in GC. DNA polymerase gamma (PolG) has been implicated in mitochondrial homeostasis and affects the development of a numerous of cancer types. However, its effects in GC and molecular mechanisms remain to be fully determined. Methods: GSE62254 dataset was used to predict the impact of PolG on prognostic valule in GC patients. Lentivirus-mediated transduction was used to silence PolG expression. Western blot analysis the knockdown effect. Co-immunoprecipitation analysis was performed to explore the potential molecular mechanism. Analysis of the glycolysis process in GC cells was also performed. Cell proliferation was determined using a CCK-8 (Cell Counting Kit-8) proliferation assay. Cell migration was detected using transwell method. Animal experiment was used to measure the In vivo xenograft tumor growth.Results: GC patients with low PolG expression have worse OS and pFS . PolG binds to PKM2 and affects the activation of the Tyr105 site phosphorylation, then interfering with the glycolysis of Gastric cancer cells. In vitro tumor formation experiments in mice also confirmed that PolG knockdown GC has stronger proliferation ability. PolG can suppress GC cells growth in both vivo and vitro.Conclusion: Our study reveals a potential molecular mechanism between PolG and the energy metabolic process of GC tumor cells, suggesting PolG as an independent novel potential therapeutic target for tumor therapy.

scholarly journals Loss of N1-methylation of G37 in tRNA induces ribosome stalling and reprograms gene expression

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Isao Masuda ◽  
Jae-Yeon Hwang ◽  
Thomas Christian ◽  
Sunita Maharjan ◽  
Fuad Mohammad ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Synthesis ◽  
Stringent Response ◽  
Peptide Bond ◽  
Ribosome Profiling ◽  
Peptide Bond Formation ◽  
Growth And Survival ◽  
Ribosome Stalling ◽  
A Genome ◽  
A Site

N1-methylation of G37 is required for a subset of tRNAs to maintain the translational reading-frame. While loss of m1G37 increases ribosomal +1 frameshifting, whether it incurs additional translational defects is unknown. Here we address this question by applying ribosome profiling to gain a genome-wide view of the effects of m1G37 deficiency on protein synthesis. Using E. coli as a model, we show that m1G37 deficiency induces ribosome stalling at codons that are normally translated by m1G37-containing tRNAs. Stalling occurs during decoding of affected codons at the ribosomal A site, indicating a distinct mechanism than that of +1 frameshifting, which occurs after the affected codons leave the A site. Enzyme- and cell-based assays show that m1G37 deficiency reduces tRNA aminoacylation and in some cases peptide-bond formation. We observe changes of gene expression in m1G37 deficiency similar to those in the stringent response that is typically induced by deficiency of amino acids. This work demonstrates a previously unrecognized function of m1G37 that emphasizes its role throughout the entire elongation cycle of protein synthesis, providing new insight into its essentiality for bacterial growth and survival.

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