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Ecotoxicology and Environmental Safety Jun 2024Environmental pollution, including air pollution, plastic contamination, and heavy metal exposure, is a pressing global issue. This crisis contributes significantly to... (Review)
Review
Environmental pollution, including air pollution, plastic contamination, and heavy metal exposure, is a pressing global issue. This crisis contributes significantly to pollution-related diseases and is a critical risk factor for chronic health conditions, including cancer. Mounting evidence underscores the pivotal role of N6-methyladenosine (mA) as a crucial regulatory mechanism in pathological processes and cancer progression. Governed by mA writers, erasers, and readers, mA orchestrates alterations in target gene expression, consequently playing a vital role in a spectrum of RNA processes, covering mRNA processing, translation, degradation, splicing, nuclear export, and folding. Thus, there is a growing need to pinpoint specific mA-regulated targets in environmental pollutant-induced carcinogenesis, an emerging area of research in cancer prevention. This review consolidates the understanding of mA modification in environmental pollutant-induced tumorigenesis, explicitly examining its implications in lung, skin, and bladder cancer. We also investigate the biological mechanisms that underlie carcinogenesis originating from pollution. Specific mA methylation pathways, such as the HIF1A/METTL3/IGF2BP3/BIRC5 network, METTL3/YTHDF1-mediated mA modification of IL 24, METTL3/YTHDF2 dynamically catalyzed mA modification of AKT1, METTL3-mediated mA-modified oxidative stress, METTL16-mediated mA modification, site-specific ATG13 methylation-mediated autophagy, and the role of mA in up-regulating ribosome biogenesis, all come into play in this intricate process. Furthermore, we discuss the direction regarding the interplay between pollutants and RNA metabolism, particularly in immune response, providing new information on RNA modifications for future exploration.
Topics: Adenosine; Carcinogenesis; Environmental Pollutants; Humans; Methylation; Animals; RNA; RNA Methylation
PubMed: 38669875
DOI: 10.1016/j.ecoenv.2024.116372 -
PeerJ 2024The propensity of nucleotide bases to form pairs, causes folding and the formation of secondary structure in the RNA. Therefore, purine (R): pyrimidine (Y) base-pairing...
INTRODUCTION
The propensity of nucleotide bases to form pairs, causes folding and the formation of secondary structure in the RNA. Therefore, purine (R): pyrimidine (Y) base-pairing is vital to maintain uniform lateral dimension in RNA secondary structure. Transversions or base substitutions between R and Y bases, are more detrimental to the stability of RNA secondary structure, than transitions derived from substitutions between A and G or C and T. The study of transversion and transition base substitutions is important to understand evolutionary mechanisms of RNA secondary structure in the 5' and 3' untranslated (UTR) regions of SARS-CoV-2. In this work, we carried out comparative analysis of transition and transversion base substitutions in the stem and loop regions of RNA secondary structure of SARS-CoV-2.
METHODS
We have considered the experimentally determined and well documented stem and loop regions of 5' and 3' UTR regions of SARS-CoV-2 for base substitution analysis. The secondary structure comprising of stem and loop regions were visualized using the RNAfold web server. The GISAID repository was used to extract base sequence alignment of the UTR regions. Python scripts were developed for comparative analysis of transversion and transition frequencies in the stem and the loop regions.
RESULTS
The results of base substitution analysis revealed a higher transition () to transversion () ratio () in the stem region of UTR of RNA secondary structure of SARS-CoV-2 reported during the early stage of the pandemic. The higher ratio in the stem region suggested the influence of secondary structure in selecting the pattern of base substitutions. This differential pattern of values between stem and loop regions was not observed among the Delta and Omicron variants that dominated the later stage of the pandemic. It is noteworthy that the values in the stem and loop regions were similar among the later dominant Delta and Omicron variant strains which is to be investigated to understand the rapid evolution and global adaptation of SARS-CoV-2.
CONCLUSION
Our findings implicate the lower frequency of transversions than the transitions in the stem regions of UTRs of SARS-CoV-2. The RNA secondary structures are associated with replication, translation, and packaging, further investigations are needed to understand these base substitutions across different variants of SARS-CoV-2.
Topics: SARS-CoV-2; RNA, Viral; Nucleic Acid Conformation; 3' Untranslated Regions; Humans; 5' Untranslated Regions; COVID-19; Base Pairing; Base Sequence
PubMed: 38666080
DOI: 10.7717/peerj.16962 -
Nucleic Acids Research Jun 2024CRISPR-Cas systems can be utilized as programmable-spectrum antimicrobials to combat bacterial infections. However, how CRISPR nucleases perform as antimicrobials across...
CRISPR-Cas systems can be utilized as programmable-spectrum antimicrobials to combat bacterial infections. However, how CRISPR nucleases perform as antimicrobials across target sites and strains remains poorly explored. Here, we address this knowledge gap by systematically interrogating the use of CRISPR antimicrobials using multidrug-resistant and hypervirulent strains of Klebsiella pneumoniae as models. Comparing different Cas nucleases, DNA-targeting nucleases outperformed RNA-targeting nucleases based on the tested targets. Focusing on AsCas12a that exhibited robust targeting across different strains, we found that the elucidated modes of escape varied widely, restraining opportunities to enhance killing. We also encountered individual guide RNAs yielding different extents of clearance across strains, which were linked to an interplay between improper gRNA folding and strain-specific DNA repair and survival. To explore features that could improve targeting across strains, we performed a genome-wide screen in different K. pneumoniae strains that yielded guide design rules and trained an algorithm for predicting guide efficiency. Finally, we showed that Cas12a antimicrobials can be exploited to eliminate K. pneumoniae when encoded in phagemids delivered by T7-like phages. Altogether, our results highlight the importance of evaluating antimicrobial activity of CRISPR antimicrobials across relevant strains and define critical parameters for efficient CRISPR-based targeting.
Topics: Klebsiella pneumoniae; CRISPR-Cas Systems; RNA, Guide, CRISPR-Cas Systems; Bacterial Proteins; Endodeoxyribonucleases; Klebsiella Infections; CRISPR-Associated Proteins; Anti-Bacterial Agents; Drug Resistance, Multiple, Bacterial; Clustered Regularly Interspaced Short Palindromic Repeats; Genome, Bacterial; Gene Editing; Humans
PubMed: 38661215
DOI: 10.1093/nar/gkae281 -
Nucleic Acids Research Jun 2024Multivalent recognition and binding of biological molecules is a natural phenomenon that increases the binding stability (avidity) without decreasing the recognition...
Multivalent recognition and binding of biological molecules is a natural phenomenon that increases the binding stability (avidity) without decreasing the recognition specificity. In this study, we took advantage of this phenomenon to increase the efficiency and maintain high specificity of RNA cleavage by DNAzymes (Dz). We designed a series of DNA constructs containing two Dz agents, named here bivalent Dz devices (BDD). One BDD increased the cleavage efficiency of a folded RNA fragment up to 17-fold in comparison with the Dz of a conventional design. Such an increase was achieved due to both the improved RNA binding and the increased probability of RNA cleavage by the two catalytic cores. By moderating the degree of Dz agent association in BDD, we achieved excellent selectivity in differentiating single-base mismatched RNA, while maintaining relatively high cleavage rates. Furthermore, a trivalent Dz demonstrated an even greater efficiency than the BDD in cleaving folded RNA. The data suggests that the cooperative action of several RNA-cleaving units can significantly improve the efficiency and maintain high specificity of RNA cleavage, which is important for the development of Dz-based gene knockdown agents.
Topics: DNA, Catalytic; Nucleic Acid Conformation; RNA; RNA Cleavage; RNA Folding; DNA; Ribonuclease H; Binding Sites; Substrate Specificity
PubMed: 38661191
DOI: 10.1093/nar/gkae295 -
ELife Apr 2024Mutations in the human gene cause the neurodevelopmental PURA syndrome. In contrast to several other monogenetic disorders, almost all reported mutations in this...
Mutations in the human gene cause the neurodevelopmental PURA syndrome. In contrast to several other monogenetic disorders, almost all reported mutations in this nucleic acid-binding protein result in the full disease penetrance. In this study, we observed that patient mutations across PURA impair its previously reported co-localization with processing bodies. These mutations either destroyed the folding integrity, RNA binding, or dimerization of PURA. We also solved the crystal structures of the N- and C-terminal PUR domains of human PURA and combined them with molecular dynamics simulations and nuclear magnetic resonance measurements. The observed unusually high dynamics and structural promiscuity of PURA indicated that this protein is particularly susceptible to mutations impairing its structural integrity. It offers an explanation why even conservative mutations across PURA result in the full penetrance of symptoms in patients with PURA syndrome.
Topics: Humans; Neurodevelopmental Disorders; Processing Bodies; Stress Granules; Crystallography, X-Ray; Dimerization; Protein Domains; Circular Dichroism; Recombinant Proteins; Protein Folding; Penetrance; Amino Acid Substitution; Point Mutation; HeLa Cells
PubMed: 38655849
DOI: 10.7554/eLife.93561 -
Frontiers in Bioengineering and... 2024Circular RNAs (circRNAs) are endogenous noncoding RNAs (ncRNAs) with transcriptional lengths ranging from hundreds to thousands. circRNAs have attracted attention owing...
INTRODUCTION
Circular RNAs (circRNAs) are endogenous noncoding RNAs (ncRNAs) with transcriptional lengths ranging from hundreds to thousands. circRNAs have attracted attention owing to their stable structure and ability to treat complicated diseases. Our objective was to create a one-step reaction for circRNA synthesis using wild-type T7 RNA polymerase as the catalyst. However, T7 RNA polymerase is thermally unstable, and we streamlined circRNA synthesis via consensus and folding free energy calculations for hotspot selection. Because of the thermal instability, the permuted intron and exon (PIE) method for circRNA synthesis is conducted via tandem catalysis with a transcription reaction at a low temperature and linear RNA precursor cyclization at a high temperature.
METHODS
To streamline the process, a multisite mutant T7 RNA polymerase (S430P, N433T, S633P, F849I, F880Y, and G788A) with significantly improved thermostability was constructed, and G788A was used.
RESULTS
The resulting mutant exhibited stable activity at 45°C for over an hour, enabling the implementation of a one-pot transcription and cyclization reaction. The simplified circRNA production process demonstrated an efficiency comparable to that of the conventional two-step reaction, with a cyclization rate exceeding 95% and reduced production of immunostimulatory dsRNA byproducts.
PubMed: 38655387
DOI: 10.3389/fbioe.2024.1356354 -
Journal of Chemical Information and... May 2024It is now known that RNAs play more active roles in cellular pathways beyond simply serving as transcription templates. These biological mechanisms might be mediated by...
It is now known that RNAs play more active roles in cellular pathways beyond simply serving as transcription templates. These biological mechanisms might be mediated by higher RNA stereo conformations, triggering the need to understand RNA secondary structures first. However, experimental protocols for solving RNA structures are unavailable for large-scale investigation due to their high costs and time-consuming nature. Various computational tools were thus developed to predict the RNA secondary structures from sequences. Recently, deep networks have been investigated to help predict RNA structures directly from their sequences. However, existing deep-learning-based tools are more or less suffering from model overfitting due to their complicated problem formulation and defective model training processes, limiting their applications across sequences from different structural families. In this research, we designed a two-stage RNA structure prediction strategy called DEBFold (deep ensemble boosting and folding) based on convolution encoding/decoding and self-attention mechanisms to enhance the existing thermodynamic structure models. Moreover, the model training process followed rigorous steps to achieve an acceptable prediction generalization. On the family-wise reserved test sets and the PDB-derived test set, DEBFold achieves better structure prediction performance over traditional tools and existing deep-learning methods. In summary, we obtained a cutting-edge deep-learning-based structure prediction tool with supreme across-family generalization performance. The DEBFold tool can be accessed at https://cobis.bme.ncku.edu.tw/DEBFold/.
Topics: Deep Learning; RNA; Nucleic Acid Conformation; Computational Biology; Models, Molecular; Thermodynamics; Base Sequence
PubMed: 38648189
DOI: 10.1021/acs.jcim.4c00458 -
Science Advances Apr 2024Nucleoprotein (NP) is a key structural protein of influenza ribonucleoprotein complexes and is central to viral RNA packing and trafficking. NP also determines the...
Nucleoprotein (NP) is a key structural protein of influenza ribonucleoprotein complexes and is central to viral RNA packing and trafficking. NP also determines the sensitivity of influenza to myxovirus resistance protein 1 (MxA), an innate immunity factor that restricts influenza replication. A few critical MxA-resistant mutations have been identified in NP, including the highly conserved proline-283 substitution. This essential proline-283 substitution impairs influenza growth, a fitness defect that becomes particularly prominent at febrile temperature (39°C) when host chaperones are depleted. Here, we biophysically characterize proline-283 NP and serine-283 NP to test whether the fitness defect is caused by the proline-283 substitution introducing folding defects. We show that the proline-283 substitution changes the folding pathway of NP, making NP more aggregation prone during folding, but does not alter the native structure of the protein. These findings suggest that influenza has evolved to hijack host chaperones to promote the folding of otherwise biophysically incompetent viral proteins that enable innate immune system escape.
Topics: Humans; Influenza, Human; Viral Core Proteins; RNA-Binding Proteins; Nucleocapsid Proteins; Myxovirus Resistance Proteins
PubMed: 38640233
DOI: 10.1126/sciadv.adl6144 -
Journal of Chemical Information and... May 2024Guanine quadruplex (GQ) is a noncanonical nucleic acid structure formed by guanine-rich DNA and RNA sequences. Folding of GQs is a complex process, where several aspects...
Guanine quadruplex (GQ) is a noncanonical nucleic acid structure formed by guanine-rich DNA and RNA sequences. Folding of GQs is a complex process, where several aspects remain elusive, despite being important for understanding structure formation and biological functions of GQs. Pulling experiments are a common tool for acquiring insights into the folding landscape of GQs. Herein, we applied a computational pulling strategy─steered molecular dynamics (SMD) simulations─in combination with standard molecular dynamics (MD) simulations to explore the unfolding landscapes of tetrameric parallel GQs. We identified anisotropic properties of elastic conformational changes, unfolding transitions, and GQ mechanical stabilities. Using a special set of structural parameters, we found that the vertical component of pulling force (perpendicular to the average G-quartet plane) plays a significant role in disrupting GQ structures and weakening their mechanical stabilities. We demonstrated that the magnitude of the vertical force component depends on the pulling anchor positions and the number of G-quartets. Typical unfolding transitions for tetrameric parallel GQs involve base unzipping, opening of the G-stem, strand slippage, and rotation to cross-like structures. The unzipping was detected as the first and dominant unfolding event, and it usually started at the 3'-end. Furthermore, results from both SMD and standard MD simulations indicate that partial spiral conformations serve as a transient ensemble during the (un)folding of GQs.
Topics: G-Quadruplexes; Molecular Dynamics Simulation; Mechanical Phenomena; Biomechanical Phenomena; DNA
PubMed: 38630447
DOI: 10.1021/acs.jcim.4c00227 -
International Journal of Molecular... Mar 2024Protein disulfide isomerase (PDI, EC 5.3.4.1) is a thiol-disulfide oxidoreductase that plays a crucial role in catalyzing the oxidation and rearrangement of disulfides...
Protein disulfide isomerase (PDI, EC 5.3.4.1) is a thiol-disulfide oxidoreductase that plays a crucial role in catalyzing the oxidation and rearrangement of disulfides in substrate proteins. In plants, PDI is primarily involved in regulating seed germination and development, facilitating the oxidative folding of storage proteins in the endosperm, and also contributing to the formation of pollen. However, the role of PDI in root growth has not been previously studied. This research investigated the impact of PDI gene deficiency in plants by using 16F16 [2-(2-Chloroacetyl)-2,3,4,9-tetrahydro-1-methyl-1H-pyrido[3,4-b]indole-1-carboxylic acid methyl ester], a small-molecule inhibitor of PDI, to remove functional redundancy. The results showed that the growth of Arabidopsis roots was significantly inhibited when treated with 16F16. To further investigate the effects of 16F16 treatment, we conducted expression profiling of treated roots using RNA sequencing and a Tandem Mass Tag (TMT)-based quantitative proteomics approach at both the transcriptomic and proteomic levels. Our analysis revealed 994 differentially expressed genes (DEGs) at the transcript level, which were predominantly enriched in pathways associated with "phenylpropane biosynthesis", "plant hormone signal transduction", "plant-pathogen interaction" and "starch and sucrose metabolism" pathways. Additionally, we identified 120 differentially expressed proteins (DEPs) at the protein level. These proteins were mainly enriched in pathways such as "phenylpropanoid biosynthesis", "photosynthesis", "biosynthesis of various plant secondary metabolites", and "biosynthesis of secondary metabolites" pathways. The comprehensive transcriptome and proteome analyses revealed a regulatory network for root shortening in Arabidopsis seedlings under 16F16 treatment, mainly involving phenylpropane biosynthesis and plant hormone signal transduction pathways. This study enhances our understanding of the significant role of PDIs in Arabidopsis root growth and provides insights into the regulatory mechanisms of root shortening following 16F16 treatment.
Topics: Protein Disulfide-Isomerases; Proteome; Transcriptome; Arabidopsis; Plant Growth Regulators; Proteomics; Carboxylic Acids; Indoles
PubMed: 38612408
DOI: 10.3390/ijms25073596