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The FEBS Journal May 2024The essential yeast protein GPN-loop GTPase 1 (Npa3) plays a critical role in RNA polymerase II (RNAPII) assembly and subsequent nuclear import. We previously identified...
The essential yeast protein GPN-loop GTPase 1 (Npa3) plays a critical role in RNA polymerase II (RNAPII) assembly and subsequent nuclear import. We previously identified a synthetic lethal interaction between a mutant lacking the carboxy-terminal 106-amino acid tail of Npa3 (npa3ΔC) and a bud27Δ mutant. As the prefoldin-like Bud27 protein participates in ribosome biogenesis and translation, we hypothesized that Npa3 may also regulate these biological processes. We investigated this proposal by using Saccharomyces cerevisiae strains episomally expressing either wild-type Npa3 or hypomorphic mutants (Npa3ΔC, Npa3K16R, and Npa3G70A). The Npa3ΔC mutant fully supports RNAPII nuclear localization and activity. However, the Npa3K16R and Npa3G70A mutants only partially mediate RNAPII nuclear targeting and exhibit a higher reduction in Npa3 function. Cell proliferation in these strains displayed an increased sensitivity to protein synthesis inhibitors hygromycin B and geneticin/G418 (npa3G70A > npa3K16R > npa3ΔC > NPA3 cells) but not to transcriptional elongation inhibitors 6-azauracil, mycophenolic acid or 1,10-phenanthroline. In all three mutant strains, the increase in sensitivity to both aminoglycoside antibiotics was totally rescued by expressing NPA3. Protein synthesis, visualized by quantifying puromycin incorporation into nascent-polypeptide chains, was markedly more sensitive to hygromycin B inhibition in npa3ΔC, npa3K16R, and npa3G70A than NPA3 cells. Notably, high-copy expression of the TIF11 gene, that encodes the eukaryotic translation initiation factor 1A (eIF1A) protein, completely suppressed both phenotypes (of reduced basal cell growth and increased sensitivity to hygromycin B) in npa3ΔC cells but not npa3K16R or npa3G70A cells. We conclude that Npa3 plays a critical RNAPII-independent and previously unrecognized role in translation initiation.
Topics: Hygromycin B; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; RNA Polymerase II; Protein Synthesis Inhibitors; GTP Phosphohydrolases; Cell Nucleus; Protein Biosynthesis
PubMed: 38431777
DOI: 10.1111/febs.17106 -
Biotechnology Journal Feb 2024Prime editing is an advanced technology in CRISPR/Cas research with increasing numbers of improved methodologies. The original multi-vector method hampers the efficiency...
Prime editing is an advanced technology in CRISPR/Cas research with increasing numbers of improved methodologies. The original multi-vector method hampers the efficiency and precision of prime editing and also has inherent difficulty in generating homozygous mutations in mammalian cells. To overcome these technical issues, we developed a Uni-vector prime editing system, wherein the major components for prime editing were constructed in all-in-one plasmids, pPE3-pPuro and pePEmax-pPuro. The Uni-vector prime editing plasmids enhance the editing efficiency of prime editing and improved the generation of homozygous mutated mammalian cell lines. The editing efficiency is dependent of the transfection efficiency. Remarkably, the Uni-vector ePE5max system achieved an impressive editing rate approximately 79% in average, even in cell lines that are traditionally difficult to transfect, such as FaDu cell line. Furthermore, it resulted in a high frequency of homozygous knocked-in cells, with a rate of 99% in HeLa and 85% in FaDu cells. Together, our Uni-vector approach simplifies the delivery of editing components and improves the editing efficiency, especially in cells with low transfection efficiency. This approach presents an advancement in the field of prime editing.
Topics: Animals; Humans; Gene Editing; HeLa Cells; Mutation; Transfection; CRISPR-Cas Systems; Mammals
PubMed: 38403398
DOI: 10.1002/biot.202300353 -
STAR Protocols Mar 2024Here, we present a protocol for lentiviral delivery of CRISPR-Cas9 to human induced pluripotent stem cell (iPSC)-derived macrophages using co-incubation with VPX...
Here, we present a protocol for lentiviral delivery of CRISPR-Cas9 to human induced pluripotent stem cell (iPSC)-derived macrophages using co-incubation with VPX virus-like particles (VPX-VLPs). We describe steps for producing polybrene and puromycin kill curves, VPX viral production, and VPX-VLP titration by western blotting. We then detail procedures for iPSC macrophage precursor lentiviral transduction and lentiviral CRISPR-Cas9-based knockout in iPSC-derived macrophages. This protocol uses efficient genome-editing techniques to explore macrophage involvement in immune response, chronic inflammation, neurodegenerative disease, and cancer progression. For complete details on the use and execution of this protocol, please refer to Navarro-Guerrero et al..
Topics: Humans; CRISPR-Cas Systems; Induced Pluripotent Stem Cells; Neurodegenerative Diseases; Gene Editing; Macrophages
PubMed: 38401123
DOI: 10.1016/j.xpro.2024.102903 -
The Journal of Biological Chemistry Mar 2024Expression of the Escherichia coli tnaCAB operon, responsible for L-tryptophan (L-Trp) transport and catabolism, is regulated by L-Trp-directed translation arrest and...
Expression of the Escherichia coli tnaCAB operon, responsible for L-tryptophan (L-Trp) transport and catabolism, is regulated by L-Trp-directed translation arrest and the ribosome arresting peptide TnaC. The function of TnaC relies on conserved residues distributed throughout the peptide, which are involved in forming an L-Trp binding site at the ribosome exit tunnel and inhibiting the ribosome function. We aimed to understand whether nonconserved amino acids surrounding these critical conserved residues play a functional role in TnaC-mediated ribosome arrest. We have isolated two intragenic suppressor mutations that restore arrest function of TnaC mutants; one of these mutations is located near the L-Trp binding site, while the other mutation is located near the ribosome active site. We used reporter gene fusions to show that both suppressor mutations have similar effects on TnaC mutants at the conserved residues involved in forming a free L-Trp binding site. However, they diverge in suppressing loss-of-function mutations in a conserved TnaC residue at the ribosome active site. With ribosome toeprinting assays, we determined that both suppressor mutations generate TnaC peptides, which are highly sensitive to L-Trp. Puromycin-challenge assays with isolated arrested ribosomes indicate that both TnaC suppressor mutants are resistant to peptidyl-tRNA cleavage by puromycin in the presence of L-Trp; however, they differ in their resistance to puromycin in the absence of L-Trp. We propose that the TnaC peptide two functionally distinct segments, a sensor domain and a stalling domain, and that the functional versatility of these domains is fine-tuned by the nature of their surrounding nonconserved residues.
Topics: Escherichia coli; Escherichia coli Proteins; Peptides; Protein Biosynthesis; Puromycin; Ribosomes
PubMed: 38395310
DOI: 10.1016/j.jbc.2024.105780 -
Bio-protocol Feb 2024As the most energy- and metabolite-consuming process, protein synthesis is under the control of several intrinsic and extrinsic factors that determine its fine-tuning to...
As the most energy- and metabolite-consuming process, protein synthesis is under the control of several intrinsic and extrinsic factors that determine its fine-tuning to the cellular microenvironment. Consequently, variations in protein synthesis rates occur under various physiological and pathological conditions, enabling an adaptive response by the cell. For example, global protein synthesis increases upon mitogenic factors to support biomass generation and cell proliferation, while exposure to low concentrations of oxygen or nutrients require translational repression and reprogramming to avoid energy depletion and cell death. To assess fluctuations in protein synthesis rates, radioactive isotopes or radiolabeled amino acids are often used. Although highly sensitive, these techniques involve the use of potentially toxic radioactive compounds and require specific materials and processes for the use and disposal of these molecules. The development of alternative, non-radioactive methods that can be easily and safely implemented in laboratories has therefore been encouraged to avoid handling radioactivity. In this context, the SUrface SEnsing of Translation (SUnSET) method, based on the classical western blot technique, was developed by Schmidt et al. in 2009. The SUnSET is nowadays recognized as a simple alternative to radioactive methods assessing protein synthesis rates. Key features • As a structural analogue of aminoacyl-transfer RNA, puromycin incorporates into the elongating peptide chain. • Detection of puromycin-labeled peptides by western blotting reflects translation rates without the need for radioactive isotopes. • The protocol described here for in vitro applications is derived from the SUnSET method originally published by Schmidt et al. (2009).
PubMed: 38379826
DOI: 10.21769/BioProtoc.4933 -
BioRxiv : the Preprint Server For... Feb 2024Loss of functional fragile X mental retardation protein (FMRP) causes fragile X syndrome (FXS) and is the leading monogenic cause of autism spectrum disorders and...
Loss of functional fragile X mental retardation protein (FMRP) causes fragile X syndrome (FXS) and is the leading monogenic cause of autism spectrum disorders and intellectual disability. FMRP is most notably a translational repressor and is thought to inhibit translation elongation by stalling ribosomes as FMRP-bound polyribosomes from brain tissue are resistant to puromycin and nuclease treatment. Here, we present data showing that the C-terminal non-canonical RNA-binding domain of FMRP is essential and sufficient to induce puromycin-resistant mRNA•ribosome complexes. Given that stalled ribosomes can stimulate ribosome collisions and no-go mRNA decay (NGD), we tested the ability of FMRP to drive NGD of its target transcripts in neuroblastoma cells. Indeed, FMRP and ribosomal proteins, but not PABPC1, were enriched in isolated nuclease-resistant disomes compared to controls. Using siRNA knockdown and RNA-seq, we identified 16 putative FMRP-mediated NGD substrates, many of which encode proteins involved in neuronal development and function. Increased mRNA stability of the putative substrates was also observed when either FMRP was depleted or NGD was prevented via RNAi. Taken together, these data support that FMRP stalls ribosomes and can stimulate NGD of a select set of transcripts in cells, revealing an unappreciated role of FMRP that would be misregulated in FXS.
PubMed: 38352534
DOI: 10.1101/2024.02.02.577121 -
International Journal of Molecular... Jan 2024Vaccinia virus () F17 protein is a major virion structural phosphoprotein having a molecular weight of 11 kDa. Recently, it was shown that F17 synthesised in infected...
Vaccinia virus () F17 protein is a major virion structural phosphoprotein having a molecular weight of 11 kDa. Recently, it was shown that F17 synthesised in infected cells interacts with mTOR subunits to evade cell immunity and stimulate late viral protein synthesis. Several years back, we purified an 11 kDa protein that inhibited protein synthesis in reticulocyte lysate from virions, and that possesses all physico-chemical properties of F17 protein. To investigate this discrepancy, we used defective vaccinia virus particles devoid of the F17 protein (designated iF17 particles) to assess their ability to inhibit protein synthesis. To this aim, we purified iF17 particles from cells infected with a vaccinia virus mutant which expresses F17 only in the presence of IPTG. The SDS-PAGE protein profiles of iF17 particles or derived particles, obtained by solubilisation of the viral membrane, were similar to that of infectious iF17 particles. As expected, the profiles of full iF17 particles and those lacking the viral membrane were missing the 11 kDa F17 band. The iF17 particles did attach to cells and injected their viral DNA into the cytoplasm. Co-infection of the non-permissive BSC40 cells with a modified vaccinia Ankara (MVA) virus, expressing an mCherry protein, and iF17 particles, induced a strong mCherry fluorescence. Altogether, these experiments confirmed that the iF17 particles can inject their content into cells. We measured the rate of protein synthesis as a function of the multiplicity of infection (MOI), in the presence of puromycin as a label. We showed that iF17 particles did not inhibit protein synthesis at high MOI, by contrast to the infectious iF17 mutant. Furthermore, the measured efficiency to inhibit protein synthesis by the iF17 mutant virus generated in the presence of IPTG, was threefold to eightfold lower than that of the wild-type WR virus. The iF17 mutant contained about threefold less F17 protein than wild-type WR. Altogether these results strongly suggest that virion-associated F17 protein is essential to mediate a stoichiometric inhibition of protein synthesis, in contrast to the late synthesised F17. It is possible that this discrepancy is due to different phosphorylation states of the free and virion-associated F17 protein.
Topics: Humans; Vaccinia virus; Vaccinia; Isopropyl Thiogalactoside; Cell Line; Phosphoproteins; Virion
PubMed: 38338659
DOI: 10.3390/ijms25031382 -
Clinical Oral Investigations Feb 2024To compare, in vitro, resin cement excess removal techniques at the veneer-tooth interface.
OBJECTIVES
To compare, in vitro, resin cement excess removal techniques at the veneer-tooth interface.
MATERIALS AND METHODS
Anterior human teeth were restored with ceramic veneers and randomly divided according to the following techniques (n = 10): removal of excess resin cement with brush and dental floss, followed by light-curing with Valo (Group 1) or Elipar (Group 2) for 1 min and 40 s; tack-curing with Valo (Group 3) or Elipar (Group 4) for 1 s; and tack-curing with Valo (Group 5) or Elipar (Group 6) for 5 s. The tack-curing was followed by removal of excess with probe and dental floss and light-curing for 1 min and 40 s. The area of excess resin cement (mm) was measured in micro-CT images using AutoCAD program. The failures at the cervical margin in the X, Y, and Z axes (µm) of greater value were measured using the DataViewer program. The specimens were submitted to microleakage with 2% basic fuchsin.
RESULTS
According to the Kruskal-Wallis and multiple comparison test, the highest area of excess resin cement was found in Group 1 (5.06 mm), which did not differ statistically from Groups 2 (3.70 mm) and 5 (2.19 mm). Groups 2, 3 (1.73 mm), 4 (1.14 mm), and 5 (2.18 mm) did not differ statistically. Group 6 (0.77 mm) obtained the lowest value, which did not differ statistically from Groups 3 and 4. According to the Kruskal-Wallis and Dunn test, there was no significant difference in failures in X (p = 0.981), Y (p = 0.860), and Z (p = 0.638) axes and no significant difference in microleakage (p = 0.203) among the groups.
CONCLUSIONS
Tack-curing for 1 s or 5 s, followed by removal of excess resin cement using a probe and a dental floss, tended to result in a lower amount of excess material around the margin.
CLINICAL RELEVANCE
The technique used for resin cement excess removal influences the amount of excess leaved at the veneer-tooth interface. Tack-curing for 1 s or 5 s is recommended to mitigate the excess resin cement.
Topics: Humans; Resin Cements; Ceramics; Neck; Puromycin; X-Ray Microtomography
PubMed: 38319457
DOI: 10.1007/s00784-024-05536-2 -
Proceedings of the National Academy of... Feb 2024Puromycin is covalently added to the nascent chain of proteins by the peptidyl transferase activity of the ribosome and the dissociation of the puromycylated peptide...
Puromycin is covalently added to the nascent chain of proteins by the peptidyl transferase activity of the ribosome and the dissociation of the puromycylated peptide typically follows this event. It was postulated that blocking the translocation of the ribosome with emetine could retain the puromycylated peptide on the ribosome, but evidence against this has recently been published [Hobson , , e60048 (2020); and Enam , , e60303 (2020)]. In neurons, puromycylated nascent chains remain in the ribosome even in the absence of emetine, yet direct evidence for this has been lacking. Using biochemistry and cryoelectron microscopy, we show that the puromycylated peptides remain in the ribosome exit channel in the large subunit in a subset of neuronal ribosomes stalled in the hybrid state. These results validate previous experiments to localize stalled polysomes in neurons and provide insight into how neuronal ribosomes are stalled. Moreover, in these hybrid-state neuronal ribosomes, anisomycin, which usually blocks puromycylation, competes poorly with puromycin in the puromycylation reaction, allowing a simple assay to determine the proportion of nascent chains that are stalled in this state. In early hippocampal neuronal cultures, over 50% of all nascent peptides are found in these stalled polysomes. These results provide insights into the stalling mechanisms of neuronal ribosomes and suggest that puromycylated peptides can be used to reveal subcellular sites of hybrid-state stalled ribosomes in neurons.
Topics: Puromycin; Cryoelectron Microscopy; Emetine; Ribosomes; Protein Biosynthesis; Peptides; Neurons
PubMed: 38315848
DOI: 10.1073/pnas.2306993121 -
Scientific Reports Feb 2024While antibiotic resistance poses a threat from both Gram-positive bacteria (GPB) and Gram-negative bacteria (GNB), GNB pose a more imminent public health hazard...
While antibiotic resistance poses a threat from both Gram-positive bacteria (GPB) and Gram-negative bacteria (GNB), GNB pose a more imminent public health hazard globally. GNB are a threat to growing antibiotic resistance because of the complex makeup of the membrane. The AcrAB-TolC efflux pump is a known resistance mechanism of Escherichia coli (E. coli) cells. This study utilized molecular dynamics modeling to visualize some of the changes occurring at a molecular level when airborne bacteria are exposed to stress and antibiotics. This study was conducted to build upon previous experimental research showing that there is an increase in antibiotic resistance and efflux pump activity when exposed to aerosolization. AcrB and AcrAB-TolC proteins were simulated under standard and increased pressure to compare the effect of aerosolization on the binding to the three different antibiotics (puromycin (PUY), ampicillin (AMP) and sulfamethoxazole-trimethoprim (SXT)) to the AcrB binding site. Analysis such as root-mean-square deviation of atomic positions and root-mean-square fluctuation, the opening of TolC, and the significant molecular mechanics with generalized Born and surface area solvation (MM-GBSA) scores associated with specific ligands were recorded. Resistance in experimental data indicated a relationship between the docking scores and some ligand-protein interactions. Results showed that there was more flexibility in the proteins within simulations conducted under standard pressure for the AcrB protein and the full tripartite complex AcrAB-TolC, showing that increased pressure causes more rigidity. MM-GBSA scores, used to calculate the free energy of ligand-protein binding, did not show a significant change, but interestingly, the strongest MM-GBSA scores were for ligands that moved to another binding pocket and did not result in resistance or opening of the efflux pump. However, the ligand moved from the binding site and did not cause the opening of TolC to increase significantly, whereas PUY and AMP were bound to the binding site for the duration of all simulations. AMP ligands under increased pressure showed the largest change in opening of the TolC efflux pump and aligns with experimental data showing E. coli cells had the most resistance to AMP after aerosolization. These results, in addition to other real-time changes such as OM proteins and mutations of targets within the cell, could be used to delineate and mitigate antibiotic resistance mechanisms.
Topics: Escherichia coli; Anti-Bacterial Agents; Escherichia coli Proteins; Membrane Transport Proteins; Molecular Dynamics Simulation; Ligands; Bacterial Outer Membrane Proteins; Multidrug Resistance-Associated Proteins; Carrier Proteins
PubMed: 38302495
DOI: 10.1038/s41598-024-52536-z