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Nucleic Acids Research Sep 2022In mammals, m7G-adjacent nucleotides undergo extensive modifications. Ribose of the first or first and second transcribed nucleotides can be subjected to...
2'-O-Methylation of the second transcribed nucleotide within the mRNA 5' cap impacts the protein production level in a cell-specific manner and contributes to RNA immune evasion.
In mammals, m7G-adjacent nucleotides undergo extensive modifications. Ribose of the first or first and second transcribed nucleotides can be subjected to 2'-O-methylation to form cap1 or cap2, respectively. When the first transcribed nucleotide is 2'-O-methylated adenosine, it can be additionally modified to N6,2'-O-dimethyladenosine (m6Am). Recently, the crucial role of cap1 in distinguishing between 'self' and 'non-self' in mammalian cells during viral infection was revealed. Here, we attempted to understand the impact of cap methylations on RNA-related processes. Therefore, we synthesized tetranucleotide cap analogues and used them for RNA capping during in vitro transcription. Using this tool, we found that 2'-O-methylation of the second transcribed nucleotide within the mRNA 5' cap influences protein production levels in a cell-specific manner. This modification can strongly hamper protein biosynthesis or have no influence on protein production levels, depending on the cell line. Interestingly, 2'-O-methylation of the second transcribed nucleotide and the presence of m6Am as the first transcribed nucleotide serve as determinants that define transcripts as 'self' and contribute to transcript escape from the host innate immune response. Additionally, cap methylation status does not influence transcript affinity towards translation initiation factor eIF4E or in vitro susceptibility to decapping by DCP2; however, we observe the resistance of cap2-RNA to DXO (decapping exoribonuclease)-mediated decapping and degradation.
Topics: Animals; Methylation; RNA, Messenger; RNA Caps; Nucleotides; Immune Evasion; Mammals
PubMed: 36018811
DOI: 10.1093/nar/gkac722 -
Journal of Molecular Biology Sep 2019Multi-subunit DNA-dependent RNA polymerases synthesize all classes of cellular RNAs, ranging from short regulatory transcripts to gigantic messenger RNAs. RNA polymerase... (Review)
Review
Multi-subunit DNA-dependent RNA polymerases synthesize all classes of cellular RNAs, ranging from short regulatory transcripts to gigantic messenger RNAs. RNA polymerase has to make each RNA product in just one try, even if it takes millions of successive nucleotide addition steps. During each step, RNA polymerase selects a correct substrate, adds it to a growing chain, and moves one nucleotide forward before repeating the cycle. However, RNA synthesis is anything but monotonous: RNA polymerase frequently pauses upon encountering mechanical, chemical and torsional barriers, sometimes stepping back and cleaving off nucleotides from the growing RNA chain. A picture in which these intermittent dynamics enable processive, accurate, and controllable RNA synthesis is emerging from complementary structural, biochemical, computational, and single-molecule studies. Here, we summarize our current understanding of the mechanism and regulation of the on-pathway transcription elongation. We review the details of substrate selection, catalysis, proofreading, and translocation, focusing on rate-limiting steps, structural elements that modulate them, and accessory proteins that appear to control RNA polymerase translocation.
Topics: DNA; DNA-Directed RNA Polymerases; Protein Binding; RNA; Ribonucleotides; Transcription Elongation, Genetic
PubMed: 31153902
DOI: 10.1016/j.jmb.2019.05.042 -
Cell Reports Oct 2022A fundamental step in regeneration is rapid growth to replace lost tissue. Cells must generate sufficient lipids, nucleotides, and proteins to fuel rapid cell division....
A fundamental step in regeneration is rapid growth to replace lost tissue. Cells must generate sufficient lipids, nucleotides, and proteins to fuel rapid cell division. To define metabolic pathways underlying regenerative growth, we undertake a multimodal investigation of metabolic reprogramming in Xenopus tropicalis appendage regeneration. Regenerating tissues have increased glucose uptake; however, inhibition of glycolysis does not decrease regeneration. Instead, glucose is funneled to the pentose phosphate pathway (PPP), which is essential for full tail regeneration. Liquid chromatography-mass spectrometry (LC-MS) metabolite profiling reveals increased nucleotide and nicotinamide intermediates required for cell division. Using single-cell RNA sequencing (scRNA-seq), we find that highly proliferative cells have increased transcription of PPP enzymes and not glycolytic enzymes. Further, PPP inhibition results in decreased cell division specifically in regenerating tissue. Our results inform a model wherein regenerating tissues direct glucose toward the PPP, yielding nucleotide precursors to drive regenerative cell proliferation.
Topics: Pentose Phosphate Pathway; Glycolysis; Glucose; Nucleotides; Niacinamide; Lipids
PubMed: 36288713
DOI: 10.1016/j.celrep.2022.111552 -
The New Phytologist Feb 2019Viruses that infect photoautotrophs have a fundamental relationship with light, given the need for host resources. We investigated the role of light on Coccolithovirus...
Viruses that infect photoautotrophs have a fundamental relationship with light, given the need for host resources. We investigated the role of light on Coccolithovirus (EhV) infection of the globally distributed coccolithophore, Emiliania huxleyi. Light was required for EhV adsorption, and viral production was highest when host cultures were maintained in continuous light or at irradiance levels of 150-300 μmol m s . During the early stages of infection, photosynthetic electron transport remained high, while RuBisCO expression decreased concomitant with an induction of the pentose phosphate pathway, the primary source of de novo nucleotides. A mathematical model developed and fitted to the laboratory data supported the hypothesis that EhV replication was controlled by a trade-off between host nucleotide recycling and de novo synthesis, and that photoperiod and photon flux could toggle this switch. Laboratory results supported field observations that light was the most robust driver of EhV replication within E. huxleyi populations collected across a 2000 nautical mile transect in the North Atlantic. Collectively, these findings demonstrate that light can drive host-virus interactions through a mechanistic interplay between host metabolic processes, which serve to structure infection and phytoplankton mortality in the upper ocean.
Topics: Adsorption; Haptophyta; Host-Pathogen Interactions; Light; NADP; Nucleotides; Pentose Phosphate Pathway; Photoperiod; Photosynthesis; Phycodnaviridae
PubMed: 30368816
DOI: 10.1111/nph.15459 -
The Journal of Biological Chemistry Apr 2023The small molecule erastin inhibits the cystine-glutamate antiporter, system x, which leads to intracellular cysteine and glutathione depletion. This can cause...
The small molecule erastin inhibits the cystine-glutamate antiporter, system x, which leads to intracellular cysteine and glutathione depletion. This can cause ferroptosis, which is an oxidative cell death process characterized by uncontrolled lipid peroxidation. Erastin and other ferroptosis inducers have been shown to affect metabolism but the metabolic effects of these drugs have not been systematically studied. To this end, we investigated how erastin impacts global metabolism in cultured cells and compared this metabolic profile to that caused by the ferroptosis inducer RAS-selective lethal 3 or in vivo cysteine deprivation. Common among the metabolic profiles were alterations in nucleotide and central carbon metabolism. Supplementing nucleosides to cysteine-deprived cells rescued cell proliferation in certain contexts, showing that these alterations to nucleotide metabolism can affect cellular fitness. While inhibition of the glutathione peroxidase GPX4 caused a similar metabolic profile as cysteine deprivation, nucleoside treatment did not rescue cell viability or proliferation under RAS-selective lethal 3 treatment, suggesting that these metabolic changes have varying importance in different scenarios of ferroptosis. Together, our study shows how global metabolism is affected during ferroptosis and points to nucleotide metabolism as an important target of cysteine deprivation.
Topics: Cell Death; Cysteine; Ferroptosis; Glutathione Peroxidase; Lipid Peroxidation; Piperazines; Nucleotides
PubMed: 36803962
DOI: 10.1016/j.jbc.2023.103039 -
International Journal of Molecular... Aug 2022Nucleotide sugars (NSs) serve as substrates for glycosylation reactions. The majority of these compounds are synthesized in the cytoplasm, whereas glycosylation occurs... (Review)
Review
Nucleotide sugars (NSs) serve as substrates for glycosylation reactions. The majority of these compounds are synthesized in the cytoplasm, whereas glycosylation occurs in the endoplasmic reticulum (ER) and Golgi lumens, where catalytic domains of glycosyltransferases (GTs) are located. Therefore, translocation of NS across the organelle membranes is a prerequisite. This process is thought to be mediated by a group of multi-transmembrane proteins from the SLC35 family, i.e., nucleotide sugar transporters (NSTs). Despite many years of research, some uncertainties/inconsistencies related with the mechanisms of NS transport and the substrate specificities of NSTs remain. Here we present a comprehensive review of the NS import into the mammalian Golgi, which consists of three major parts. In the first part, we provide a historical view of the experimental approaches used to study NS transport and evaluate the most important achievements. The second part summarizes various aspects of knowledge concerning NSTs, ranging from subcellular localization up to the pathologies related with their defective function. In the third part, we present the outcomes of our research performed using mammalian cell-based models and discuss its relevance in relation to the general context.
Topics: Animals; Biological Transport; Glycosylation; Golgi Apparatus; Mammals; Nucleotides; Sugars
PubMed: 35955785
DOI: 10.3390/ijms23158648 -
Scientific Reports Aug 2021Gold nanoparticles (AuNPs) decorated with biologically relevant molecules have variety of applications in optical sensing of bioanalytes. Coating AuNPs with small...
Gold nanoparticles (AuNPs) decorated with biologically relevant molecules have variety of applications in optical sensing of bioanalytes. Coating AuNPs with small nucleotides produces particles with high stability in water, but functionality-compatible strategies are needed to uncover the full potential of this type of conjugates. Here, we demonstrate that lipoic acid-modified dinucleotides can be used to modify AuNPs surfaces in a controllable manner to produce conjugates that are stable in aqueous buffers and biological mixtures and capable of interacting with nucleotide-binding proteins. Using this strategy we obtained AuNPs decorated with 7-methylguanosine mRNA 5' cap analogs and showed that they bind cap-specific protein, eIF4E. AuNPs decorated with non-functional dinucleotides also interacted with eIF4E, albeit with lower affinity, suggesting that eIF4E binding to cap-decorated AuNPs is partially mediated by unspecific ionic interactions. This issue was overcome by applying lipoic-acid-Tris conjugate as a charge-neutral diluting molecule. Tris-Lipo-diluted cap-AuNPs conjugates interacted with eIF4E in fully specific manner, enabling design of functional tools. To demonstrate the potential of these conjugates in protein sensing, we designed a two-component eIF4E sensing system consisting of cap-AuNP and 4E-BP1-AuNP conjugates, wherein 4E-BP1 is a short peptide derived from 4E-BP protein that specifically binds eIF4E at a site different to that of the 5' cap. This system facilitated controlled aggregation, in which eIF4E plays the role of the agent that crosslinks two types of AuNP, thereby inducing a naked-eye visible absorbance redshift. The reported AuNPs-nucleotide conjugation method based on lipoic acid affinity for gold, can be harnessed to obtain other types of nucleotide-functionalized AuNPs, thereby paving the way to studying other nucleotide-binding proteins.
Topics: Adaptor Proteins, Signal Transducing; Biosensing Techniques; Carrier Proteins; Cell Cycle Proteins; Gold; Humans; Metal Nanoparticles; Nucleotides; Peptide Fragments; Protein Binding; Protein Biosynthesis; RNA Caps
PubMed: 34344911
DOI: 10.1038/s41598-021-94983-y -
Communications Biology Oct 2023The respiratory syncytial virus polymerase complex, consisting of the polymerase (L) and phosphoprotein (P), catalyzes nucleotide polymerization, cap addition, and cap...
The respiratory syncytial virus polymerase complex, consisting of the polymerase (L) and phosphoprotein (P), catalyzes nucleotide polymerization, cap addition, and cap methylation via the RNA dependent RNA polymerase, capping, and Methyltransferase domains on L. Several nucleoside and non-nucleoside inhibitors have been reported to inhibit this polymerase complex, but the structural details of the exact inhibitor-polymerase interactions have been lacking. Here, we report a non-nucleoside inhibitor JNJ-8003 with sub-nanomolar inhibition potency in both antiviral and polymerase assays. Our 2.9 Å resolution cryo-EM structure revealed that JNJ-8003 binds to an induced-fit pocket on the capping domain, with multiple interactions consistent with its tight binding and resistance mutation profile. The minigenome and gel-based de novo RNA synthesis and primer extension assays demonstrated that JNJ-8003 inhibited nucleotide polymerization at the early stages of RNA transcription and replication. Our results support that JNJ-8003 binding modulates a functional interplay between the capping and RdRp domains, and this molecular insight could accelerate the design of broad-spectrum antiviral drugs.
Topics: Respiratory Syncytial Virus, Human; RNA-Dependent RNA Polymerase; Protein Binding; RNA; Nucleotides
PubMed: 37865687
DOI: 10.1038/s42003-023-05451-4 -
Chemical Research in Toxicology Jan 2021The base and nucleotide excision repair pathways (BER and NER, respectively) are two major mechanisms that remove DNA lesions formed by the reactions of genotoxic...
The base and nucleotide excision repair pathways (BER and NER, respectively) are two major mechanisms that remove DNA lesions formed by the reactions of genotoxic intermediates with cellular DNA. We have demonstrated earlier that the oxidatively generated guanine lesions spiroiminodihydantoin (Sp) and 5-guanidinohydantoin (Gh) are excised from double-stranded DNA by competing BER and NER in whole-cell extracts [Shafirovich, V., et al. (2016) . , 5309-5319]. In this work we compared the NER and BER yields with single Gh or Sp lesions embedded at the same sites in covalently closed circular pUC19NN plasmid DNA (cccDNA) and in the same but linearized form (linDNA) of this plasmid. The kinetics of the Sp and Gh BER and NER incisions were monitored in HeLa cell extracts. The yield of NER products is ∼5 times greater in covalently closed circular DNA than in the linearized form, while the BER yield is smaller by ∼20-30% depending on the guanine lesion. Control BER experiments with 8-oxo-7,8-dihydroguanine (8-oxoG) show that the BER yield is increased by a factor of only 1.4 ± 0.2 in cccDNA relative to linDNA. These surprising differences in BER and NER activities are discussed in terms of the lack of termini in covalently closed circular DNA and the DNA lesion search dynamics of the NER DNA damage sensor XPC-RAD23B and the BER enzyme OGG1 that recognizes and excises 8-oxoG.
Topics: DNA; DNA Repair; Guanine; HeLa Cells; Humans; Nucleic Acid Conformation; Nucleotides; Oxidation-Reduction; Plasmids
PubMed: 33405911
DOI: 10.1021/acs.chemrestox.0c00463 -
The Journal of Biological Chemistry Jul 2022The nucleotide context surrounding stop codons significantly affects the efficiency of translation termination. In eukaryotes, various 3' contexts that are unfavorable...
The nucleotide context surrounding stop codons significantly affects the efficiency of translation termination. In eukaryotes, various 3' contexts that are unfavorable for translation termination have been described; however, the exact molecular mechanism that mediates their effects remains unknown. In this study, we used a reconstituted mammalian translation system to examine the efficiency of stop codons in different contexts, including several previously described weak 3' stop codon contexts. We developed an approach to estimate the level of stop codon readthrough in the absence of eukaryotic release factors (eRFs). In this system, the stop codon is recognized by the suppressor or near-cognate tRNAs. We observed that in the absence of eRFs, readthrough occurs in a 3' nucleotide context-dependent manner, and the main factors determining readthrough efficiency were the type of stop codon and the sequence of the 3' nucleotides. Moreover, the efficiency of translation termination in weak 3' contexts was almost equal to that in the tested standard context. Therefore, the ability of eRFs to recognize stop codons and induce peptide release is not affected by mRNA context. We propose that ribosomes or other participants of the elongation cycle can independently recognize certain contexts and increase the readthrough of stop codons. Thus, the efficiency of translation termination is regulated by the 3' nucleotide context following the stop codon and depends on the concentrations of eRFs and suppressor/near-cognate tRNAs.
Topics: Animals; Codon, Terminator; Eukaryota; Humans; Mammals; Nucleotides; Peptide Chain Elongation, Translational; Peptide Chain Termination, Translational; Peptide Termination Factors; Protein Biosynthesis; RNA, Transfer; Ribosomes
PubMed: 35700825
DOI: 10.1016/j.jbc.2022.102133