16,279 results on '"Base sequence"'
Search Results
2. Dynamics of cis -regulatory sequences and transcriptional divergence of duplicated genes in soybean.
- Author
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Fang C, Yang M, Tang Y, Zhang L, Zhao H, Ni H, Chen Q, Meng F, and Jiang J
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- Genome, Genes, Duplicate genetics, Base Sequence, Gene Duplication, Genome, Plant genetics, Glycine max genetics, Glycine max metabolism, Evolution, Molecular
- Abstract
Transcriptional divergence of duplicated genes after whole genome duplication (WGD) has been described in many plant lineages and is often associated with subgenome dominance, a genome-wide mechanism. However, it is unknown what underlies the transcriptional divergence of duplicated genes in polyploid species that lack subgenome dominance. Soybean is a paleotetraploid with a WGD that occurred 5 to 13 Mya. Approximately 50% of the duplicated genes retained from this WGD exhibit transcriptional divergence. We developed accessible chromatin region (ACR) datasets from leaf, flower, and seed tissues using MNase-hypersensitivity sequencing. We validated enhancer function of several ACRs associated with known genes using CRISPR/Cas9-mediated genome editing. The ACR datasets were used to examine and correlate the transcriptional patterns of 17,111 pairs of duplicated genes in different tissues. We demonstrate that ACR dynamics are correlated with divergence of both expression level and tissue specificity of individual gene pairs. Gain or loss of flanking ACRs and mutation of cis -regulatory elements (CREs) within the ACRs can change the balance of the expression level and/or tissue specificity of the duplicated genes. Analysis of DNA sequences associated with ACRs revealed that the extensive sequence rearrangement after the WGD reshaped the CRE landscape, which appears to play a key role in the transcriptional divergence of duplicated genes in soybean. This may represent a general mechanism for transcriptional divergence of duplicated genes in polyploids that lack subgenome dominance.
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- 2023
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3. Pervasive RNA folding is crucial for narnavirus genome maintenance.
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Fukuda M, Cai J, Bader JS, and Boeke JD
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- RNA Folding, Genome, Viral genetics, Base Sequence, Replicon genetics, Virus Replication, RNA, Viral genetics, RNA, Viral chemistry, RNA Viruses genetics
- Abstract
A synthetic biology approach toward constructing an RNA-based genome expands our understanding of living things and opens avenues for technological advancement. For the precise design of an artificial RNA replicon either from scratch or based on a natural RNA replicon, understanding structure-function relationships of RNA sequences is critical. However, our knowledge remains limited to a few particular structural elements intensively studied so far. Here, we conducted a series of site-directed mutagenesis studies of yeast narnaviruses ScNV20S and ScNV23S, perhaps the simplest natural autonomous RNA replicons, to identify RNA elements required for maintenance and replication. RNA structure disruption corresponding to various portions of the entire narnavirus genome suggests that pervasive RNA folding, in addition to the precise secondary structure of genome termini, is essential for maintenance of the RNA replicon in vivo. Computational RNA structure analyses suggest that this scenario likely applies to other "narna-like" viruses. This finding implies selective pressure on these simplest autonomous natural RNA replicons to fold into a unique structure that acquires both thermodynamic and biological stability. We propose the importance of pervasive RNA folding for the design of RNA replicons that could serve as a platform for in vivo continuous evolution as well as an interesting model to study the origin of life.
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- 2023
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4. In vivo mRNA display enables large-scale proteomics by next generation sequencing
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Saeed Tavazoie, Roberto Salatino, and Panos Oikonomou
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Proteomics ,Saccharomyces cerevisiae Proteins ,Proteome ,Context (language use) ,Computational biology ,protein–protein interactions ,Biology ,DNA sequencing ,Protein–protein interaction ,Protein Interaction Mapping ,mRNA display ,RNA, Messenger ,DNA, Fungal ,protein display technologies ,Gene Library ,Multidisciplinary ,Base Sequence ,RNA ,High-Throughput Nucleotide Sequencing ,Sequence Analysis, DNA ,Biological Sciences ,Applied Biological Sciences ,Binding domain ,MS2 tagging - Abstract
Significance The need for mass-spectrometric determination of protein identity severely limits the throughput of many whole-cell proteomic analyses. We have developed an approach to physically connect in vivo-expressed proteins to their encoding messenger RNAs (mRNAs). This genotype–phenotype linkage enables us to carry out a variety of whole-proteome assays and determine the identity of each protein using nucleic acid sequencing. We show that the resulting in vivo mRNA display technology enables determination of protein subcellular localization and protein–protein interactions, with high sensitivity and specificity, using simple purification followed by high-throughput sequencing. In vivo mRNA display should enable more efficient interrogation of proteome behavior in a variety of applications that are currently limited by the cost and throughput of mass-spectrometric analysis., Large-scale proteomic methods are essential for the functional characterization of proteins in their native cellular context. However, proteomics has lagged far behind genomic approaches in scalability, standardization, and cost. Here, we introduce in vivo mRNA display, a technology that converts a variety of proteomics applications into a DNA sequencing problem. In vivo-expressed proteins are coupled with their encoding messenger RNAs (mRNAs) via a high-affinity stem-loop RNA binding domain interaction, enabling high-throughput identification of proteins with high sensitivity and specificity by next generation DNA sequencing. We have generated a high-coverage in vivo mRNA display library of the Saccharomyces cerevisiae proteome and demonstrated its potential for characterizing subcellular localization and interactions of proteins expressed in their native cellular context. In vivo mRNA display libraries promise to circumvent the limitations of mass spectrometry-based proteomics and leverage the exponentially improving cost and throughput of DNA sequencing to systematically characterize native functional proteomes.
- Published
- 2020
5. Standards Recommendations for the Earth BioGenome Project
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Mara K. N. Lawniczak, Richard Durbin, Paul Flicek, Kerstin Lindblad-Toh, Xiaofeng Wei, John M. Archibald, William J. Baker, Katherine Belov, Mark L. Blaxter, Tomas Marques Bonet, Anna K. Childers, Jonathan A. Coddington, Keith A. Crandall, Andrew J. Crawford, Robert P. Davey, Federica Di Palma, Qi Fang, Wilfried Haerty, Neil Hall, Katharina J. Hoff, Kerstin Howe, Erich D. Jarvis, Warren E. Johnson, Rebecca N. Johnson, Paul J. Kersey, Xin Liu, Jose Victor Lopez, Eugene W. Myers, Olga Vinnere Pettersson, Adam M. Phillippy, Monica F. Poelchau, Kim D. Pruitt, Arang Rhie, Juan Carlos Castilla-Rubio, Sunil Kumar Sahu, Nicholas A. Salmon, Pamela S. Soltis, David Swarbreck, Françoise Thibaud-Nissen, Sibo Wang, Jill L. Wegrzyn, Guojie Zhang, He Zhang, Harris A. Lewin, Stephen Richards, Durbin, Richard [0000-0002-9130-1006], Apollo - University of Cambridge Repository, National Library of Medicine (US), National Institutes of Health (US), Swedish Research Council, National Museum of Natural History Smithsonian Institution, Howard Hughes Medical Institute, Wellcome, European Molecular Biology Laboratory, National Science Foundation (US), Department of Agriculture (US), and Agricultural Research Service (US)
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Evolution ,Earth BioGenome Project ,Evolutionsbiologi ,Reference Values ,Genetics ,genomics ,Animals ,Humans ,The Earth BioGenome Project: The Launch of a Moonshot for Biology ,Ethics ,Evolutionary Biology ,Multidisciplinary ,Genome assembly ,Base Sequence ,Human Genome ,Eukaryota ,DNA ,Genomics ,Biodiversity ,Sequence Analysis, DNA ,Biological Sciences ,Reference Standards ,ethics ,Perspective ,genome assembly ,Sequence Analysis - Abstract
Funder: Howard Hughes Medical Institute, Funder: National Science Foundation; Grant(s): DBI:IIBR:CAREER #1943371, A global international initiative such as the Earth BioGenome Project (EBP) requires both agreement and coordination on standards to ensure that the collective effort generates rapid progress towards its goals. To this end, the EBP initiated five technical standards committees comprising volunteer members from the global genomics scientific community: Sample Collection and Processing, Sequencing and Assembly, Annotation, Analysis, and, IT and Informatics. The current versions of the resulting standards documents are available on the EBP website, with the recognition that opportunities, technologies and challenges may improve or change in the future requiring flexibility for the EBP to meet its goals. Here, we describe some highlights from the proposed standards, and areas where additional challenges will need to be met.
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- 2022
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6. RNA polymerase II trapped on a molecular treadmill: Structural basis of persistent transcriptional arrest by a minor groove DNA binder
- Author
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Juntaek Oh, Tiezheng Jia, Jun Xu, Jenny Chong, Peter B. Dervan, and Dong Wang
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Models, Molecular ,Multidisciplinary ,Base Sequence ,Transcription, Genetic ,Imidazoles ,Nucleic Acid Conformation ,Pyrroles ,DNA ,RNA Polymerase II ,Transcriptional Elongation Factors - Abstract
Elongating RNA polymerase II (Pol II) can be paused or arrested by a variety of obstacles. These obstacles include DNA lesions, DNA-binding proteins, and small molecules. Hairpin pyrrole-imidazole (Py-Im) polyamides bind to the minor groove of DNA in a sequence-specific manner and induce strong transcriptional arrest. Remarkably, this Py-Im–induced Pol II transcriptional arrest is persistent and cannot be rescued by transcription factor TFIIS. In contrast, TFIIS can effectively rescue the transcriptional arrest induced by a nucleosome barrier. The structural basis of Py-Im–induced transcriptional arrest and why TFIIS cannot rescue this arrest remain elusive. Here we determined the X-ray crystal structures of four distinct Pol II elongation complexes (Pol II ECs) in complex with hairpin Py-Im polyamides as well as of the hairpin Py-Im polyamides–dsDNA complex. We observed that the Py-Im oligomer directly interacts with RNA Pol II residues, introduces compression of the downstream DNA duplex, prevents Pol II forward translocation, and induces Pol II backtracking. These results, together with biochemical studies, provide structural insight into the molecular mechanism by which Py-Im blocks transcription. Our structural study reveals why TFIIS fails to promote Pol II bypass of Py-Im–induced transcriptional arrest.
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- 2022
7. Efficient precise integration of large DNA sequences with 3'-overhang dsDNA donors using CRISPR/Cas9.
- Author
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Han W, Li Z, Guo Y, He K, Li W, Xu C, Ge L, He M, Yin X, Zhou J, Li C, Yao D, Bao J, and Liang H
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- Animals, Base Sequence, DNA genetics, Homologous Recombination, Mammals genetics, CRISPR-Cas Systems genetics, Gene Editing methods
- Abstract
CRISPR/Cas9 genome-editing tools have tremendously boosted our capability of manipulating the eukaryotic genomes in biomedical research and innovative biotechnologies. However, the current approaches that allow precise integration of gene-sized large DNA fragments generally suffer from low efficiency and high cost. Herein, we developed a versatile and efficient approach, termed LOCK ( L ong dsDNA with 3'- O verhangs mediated C RISPR K nock-in), by utilizing specially designed 3'-overhang double-stranded DNA (odsDNA) donors harboring 50-nt homology arm. The length of the 3'-overhangs of odsDNA is specified by the five consecutive phosphorothioate modifications. Compared with existing methods, LOCK allows highly efficient targeted insertion of kilobase-sized DNA fragments into the mammalian genomes with low cost and low off-target effects, yielding >fivefold higher knock-in frequencies than conventional homologous recombination-based approaches. This newly designed LOCK approach based on homology-directed repair is a powerful tool suitable for gene-sized fragment integration that is urgently needed for genetic engineering, gene therapies, and synthetic biology.
- Published
- 2023
- Full Text
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8. Nontriplet feature of genetic code in Euplotes ciliates is a result of neutral evolution.
- Author
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Gaydukova SA, Moldovan MA, Vallesi A, Heaphy SM, Atkins JF, Gelfand MS, and Baranov PV
- Subjects
- Genetic Code, Base Sequence, Codon, Terminator genetics, Codon, Terminator metabolism, Genetic Drift, Euplotes genetics, Euplotes metabolism, Ciliophora genetics
- Abstract
The triplet nature of the genetic code is considered a universal feature of known organisms. However, frequent stop codons at internal mRNA positions in Euplotes ciliates ultimately specify ribosomal frameshifting by one or two nucleotides depending on the context, thus posing a nontriplet feature of the genetic code of these organisms. Here, we sequenced transcriptomes of eight Euplotes species and assessed evolutionary patterns arising at frameshift sites. We show that frameshift sites are currently accumulating more rapidly by genetic drift than they are removed by weak selection. The time needed to reach the mutational equilibrium is several times longer than the age of Euplotes and is expected to occur after a several-fold increase in the frequency of frameshift sites. This suggests that Euplotes are at an early stage of the spread of frameshifting in expression of their genome. In addition, we find the net fitness burden of frameshift sites to be noncritical for the survival of Euplotes . Our results suggest that fundamental genome-wide changes such as a violation of the triplet character of genetic code can be introduced and maintained solely by neutral evolution.
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- 2023
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9. Evolution of coronavirus frameshifting elements: Competing stem networks explain conservation and variability.
- Author
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Yan S, Zhu Q, Hohl J, Dong A, and Schlick T
- Subjects
- Humans, RNA, Viral metabolism, Base Sequence, Nucleic Acid Conformation, Frameshifting, Ribosomal genetics, Coronavirus genetics, Coronavirus metabolism, Coronavirus Infections genetics
- Abstract
The frameshifting RNA element (FSE) in coronaviruses (CoVs) regulates the programmed -1 ribosomal frameshift (-1 PRF) mechanism common to many viruses. The FSE is of particular interest as a promising drug candidate. Its associated pseudoknot or stem loop structure is thought to play a large role in frameshifting and thus viral protein production. To investigate the FSE structural evolution, we use our graph theory-based methods for representing RNA secondary structures in the RNA-As-Graphs (RAG) framework to calculate conformational landscapes of viral FSEs with increasing sequence lengths for representative 10 Alpha and 13 Beta-CoVs. By following length-dependent conformational changes, we show that FSE sequences encode many possible competing stems which in turn favor certain FSE topologies, including a variety of pseudoknots, stem loops, and junctions. We explain alternative competing stems and topological FSE changes by recurring patterns of mutations. At the same time, FSE topology robustness can be understood by shifted stems within different sequence contexts and base pair coevolution. We further propose that the topology changes reflected by length-dependent conformations contribute to tuning the frameshifting efficiency. Our work provides tools to analyze virus sequence/structure correlations, explains how sequence and FSE structure have evolved for CoVs, and provides insights into potential mutations for therapeutic applications against a broad spectrum of CoV FSEs by targeting key sequence/structural transitions.
- Published
- 2023
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10. A mutant fitness assay identifies bacterial interactions in a model ocean hot spot.
- Author
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Schreier JE, Smith CB, Ioerger TR, and Moran MA
- Subjects
- Phytoplankton metabolism, Base Sequence, Oceans and Seas, Seawater microbiology, Diatoms genetics
- Abstract
Bacteria that assemble in phycospheres surrounding living phytoplankton cells metabolize a substantial proportion of ocean primary productivity. Yet the type and extent of interactions occurring among species that colonize these micron-scale "hot spot" environments are challenging to study. We identified genes that mediate bacterial interactions in phycosphere communities by culturing a transposon mutant library of copiotrophic bacterium Ruegeria pomeroyi DSS-3 with the diatom Thalassiosira pseudonana CCMP1335 as the sole source of organic matter in the presence or absence of other heterotrophic bacterial species. The function of genes having significant effects on R. pomeroyi fitness indicated explicit cell-cell interactions initiated in the multibacterial phycospheres. We found that R. pomeroyi simultaneously competed for shared substrates while increasing reliance on substrates that did not support the other species' growth. Fitness outcomes also indicated that the bacterium competed for nitrogen in the forms of ammonium and amino acids; obtained purines, pyrimidines, and cofactors via crossfeeding; both initiated and defended antagonistic interactions; and sensed an environment with altered oxygen and superoxide levels. The large genomes characteristic of copiotrophic marine bacteria are hypothesized to enable responses to dynamic ecological challenges occurring at the scale of microns. Here, we discover >200 nonessential genes implicated in the management of fitness costs and benefits of membership in a globally significant bacterial community.
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- 2023
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11. Identification of the initial nucleocapsid recognition element in the HIV-1 RNA packaging signal
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Aaron Kidane, Emily Cannistraci, Heather M. Frank, Hana Flores, Alice Telesnitsky, Christina Quasney, Pengfei Ding, Verna Van, Alexis Waller, Sapna Basappa, Nansen Kuo, Ugonna Mbaekwe, Michael F. Summers, Canessa Swanson, Siarhei Kharytonchyk, Mitali Sarkar, and Ridhi Chaudhary
- Subjects
Base pair ,packaging ,nucleocapsid ,HIV Infections ,Genome, Viral ,Regulatory Sequences, Ribonucleic Acid ,Biochemistry ,Hiv 1 rna ,Virus ,Commentaries ,Humans ,Binding site ,genome ,Binding Sites ,Multidisciplinary ,Base Sequence ,Chemistry ,Virus Assembly ,Virion ,RNA ,Nucleocapsid Proteins ,Biological Sciences ,Affinities ,Helix ,Biophysics ,HIV-1 ,Nucleic Acid Conformation ,RNA, Viral ,Function (biology) ,Protein Binding - Abstract
Significance Understanding the molecular determinants of retroviral genome packaging is important for drug discovery and development of vectors for gene delivery. We show that the HIV-1 leader, which contains the RNA elements necessary for genome packaging, binds approximately two dozen copies of the cognate NC protein with affinities ranging from ∼40 nM to 1.4 µM. Binding to the four highest-affinity “initial” binding sites occurs with endothermic energetics attributed to NC-induced localized RNA melting. Mutations that stabilize these sites inhibit NC binding in vitro and RNA packaging in transfected cells. A small-molecule inhibitor of RNA packaging binds specifically to the initial NC binding sites and stabilizes the RNA structure. Our findings identify a potential RNA Achilles’ heel for HIV therapeutic development., Selective packaging of the HIV-1 genome during virus assembly is mediated by interactions between the dimeric 5ʹ-leader of the unspliced viral RNA and the nucleocapsid (NC) domains of a small number of assembling viral Gag polyproteins. Here, we show that the dimeric 5′-leader contains more than two dozen NC binding sites with affinities ranging from 40 nM to 1.4 μM, and that all high-affinity sites (Kd ≲ 400 nM) reside within a ∼150-nt region of the leader sufficient to promote RNA packaging (core encapsidation signal, ΨCES). The four initial binding sites with highest affinity reside near two symmetrically equivalent three-way junction structures. Unlike the other high-affinity sites, which bind NC with exothermic energetics, binding to these sites occurs endothermically due to concomitant unwinding of a weakly base-paired [UUUU]:[GGAG] helical element. Mutations that stabilize base pairing within this element eliminate NC binding to this site and severely impair RNA packaging into virus-like particles. NMR studies reveal that a recently discovered small-molecule inhibitor of HIV-1 RNA packaging that appears to function by stabilizing the structure of the leader binds directly to the [UUUU]:[GGAG] helix. Our findings suggest a sequential NC binding mechanism for Gag-genome assembly and identify a potential RNA Achilles’ heel to which HIV therapeutics may be targeted.
- Published
- 2020
12. Identification of U11snRNA as an endogenous agonist of TLR7-mediated immune pathogenesis
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Tohru Natsume, Nobuyasu Endo, Shunji Takemura, Takaya Abe, Hiroyuki Ishiwata, Takako Negishi-Koga, Tadatsugu Taniguchi, Ryuji Koshiba, Tomohisa Okamura, Shun-ichiro Iemura, Takeshi Ichinohe, Kosuke Matsuki, Keishi Fujio, Hiroshi Kiyonari, Yuki Nakajima, Hideo Negishi, Junko Nishio, Kazuhiko Yamamoto, Tatsuaki Nishiyama, Takeshi Doi, Atsushi Matsuda, Hideyuki Yanai, Yuichiro Tabunoki, and Sho Hangai
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Damp ,Adult ,Biology ,medicine.disease_cause ,Autoimmunity ,Autoimmune Diseases ,Pathogenesis ,Arthritis, Rheumatoid ,Mice ,Young Adult ,Immune system ,Immunology and Inflammation ,U11snRNA ,Cell Line, Tumor ,RNA, Small Nuclear ,medicine ,Alarmins ,Animals ,Humans ,Lupus Erythematosus, Systemic ,Receptor ,TLR7 ,DAMPs ,Mice, Inbred BALB C ,Multidisciplinary ,Innate immune system ,Membrane Glycoproteins ,Base Sequence ,Sequence Analysis, RNA ,RNA ,virus diseases ,Biological Sciences ,Middle Aged ,Ribonucleoproteins, Small Nuclear ,Mice, Inbred C57BL ,Disease Models, Animal ,Toll-Like Receptor 7 ,Mice, Inbred DBA ,Immunology ,Female ,type I IFN ,Immunosuppressive Agents - Abstract
Significance Immunogenic damage-associated molecular patterns (DAMPs), typically released from dying cells, can evoke chronic inflammatory or autoimmune disorders via their activation of innate immune receptors. Since an association of RNA-sensing Toll-like receptor 7 (TLR7) signaling with autoimmunity is well-documented, identification of a DAMP(s) that triggers TLR7 is critical to understanding the disease pathogenesis. By generating the synthetic compound KN69 that inhibits autoimmunity in mouse models, we identified U11 small nuclear RNA (U11snRNA) as a target of KN69 and strong activator of TLR7. We found a correlation between high serum level of U11snRNA and autoimmune diseases in human subjects and mouse models. Finally, we generated TLR7 agonists and TLR7 antagonists. Our study provides therapeutic insight into autoimmunity and other diseases., The activation of innate immune receptors by pathogen-associated molecular patterns (PAMPs) is central to host defense against infections. On the other hand, these receptors are also activated by immunogenic damage-associated molecular patterns (DAMPs), typically released from dying cells, and the activation can evoke chronic inflammatory or autoimmune disorders. One of the best known receptors involved in the immune pathogenesis is Toll-like receptor 7 (TLR7), which recognizes RNA with single-stranded structure. However, the causative DAMP RNA(s) in the pathogenesis has yet to be identified. Here, we first developed a chemical compound, termed KN69, that suppresses autoimmunity in several established mouse models. A subsequent search for KN69-binding partners led to the identification of U11 small nuclear RNA (U11snRNA) as a candidate DAMP RNA involved in TLR7-induced autoimmunity. We then showed that U11snRNA robustly activated the TLR7 pathway in vitro and induced arthritis disease in vivo. We also found a correlation between high serum level of U11snRNA and autoimmune diseases in human subjects and established mouse models. Finally, by revealing the structural basis for U11snRNA’s ability to activate TLR7, we developed more potent TLR7 agonists and TLR7 antagonists, which may offer new therapeutic approaches for autoimmunity or other immune-driven diseases. Thus, our study has revealed a hitherto unknown immune function of U11snRNA, providing insight into TLR7-mediated autoimmunity and its potential for further therapeutic applications.
- Published
- 2019
13. Asymmetric base-pair opening drives helicase unwinding dynamics
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Yaakov Levy, Francesco Colizzi, Remi Fritzen, Cibran Perez-Gonzalez, Malcolm F. White, Giovanni Bussi, J. Carlos Penedo, The Wellcome Trust, University of St Andrews. School of Physics and Astronomy, University of St Andrews. Biomedical Sciences Research Complex, University of St Andrews. School of Biology, and University of St Andrews. Centre for Biophotonics
- Subjects
Double helix ,Experiments ,Nucleic acids ,Simulations ,Unwindability ,QH301 Biology ,01 natural sciences ,Biochemistry ,Nucleobase ,chemistry.chemical_compound ,Transcription (biology) ,Base Pairing ,R2C ,0303 health sciences ,Multidisciplinary ,010304 chemical physics ,biology ,Chemistry ,experiments ,Biological Sciences ,nucleic acids ,RNA, Bacterial ,Physical Sciences ,simulations ,BDC ,DNA, Bacterial ,Base pair ,double helix ,QH426 Genetics ,Settore FIS/03 - Fisica della Materia ,QH301 ,03 medical and health sciences ,Bacterial Proteins ,0103 physical sciences ,QH426 ,030304 developmental biology ,Bacteria ,Base Sequence ,DNA Helicases ,RNA ,Helicase ,DAS ,Biophysics and Computational Biology ,Kinetics ,unwindability ,Duplex (building) ,Biophysics ,biology.protein ,Nucleic acid ,DNA - Abstract
Significance Six decades after DNA structure was first revealed, fundamental questions remain open. How is the entwined embrace of double-stranded nucleic acids formed or disrupted? How does the energetics underlying this process influence nucleic-acid processing machineries? By combining simulations and experiments, our work addresses these questions and reveals that asymmetric base-pair dynamics drives the stepwise separation of nucleic acid duplexes, predicts the unwinding efficiency of helicases, and intimately relates the intrinsic dynamics of base pairs to the enzymatic mechanism evolved for their opening. Taken together, our data suggest a layer of regulation of the genetic material encoded in the “unwindability” of the double helix., The opening of a Watson–Crick double helix is required for crucial cellular processes, including replication, repair, and transcription. It has long been assumed that RNA or DNA base pairs are broken by the concerted symmetric movement of complementary nucleobases. By analyzing thousands of base-pair opening and closing events from molecular simulations, here, we uncover a systematic stepwise process driven by the asymmetric flipping-out probability of paired nucleobases. We demonstrate experimentally that such asymmetry strongly biases the unwinding efficiency of DNA helicases toward substrates that bear highly dynamic nucleobases, such as pyrimidines, on the displaced strand. Duplex substrates with identical thermodynamic stability are thus shown to be more easily unwound from one side than the other, in a quantifiable and predictable manner. Our results indicate a possible layer of gene regulation coded in the direction-dependent unwindability of the double helix.
- Published
- 2019
14. Ciliopathy genes are required for apical secretion of Cochlin, an otolith crystallization factor
- Author
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Zhou Zhu, Eleanor Markham, Fredericus J.M. van Eeden, Robert A. Hirst, Jarema Malicki, Xiaoming Fang, Eleni Leventea, and Yulia Nikolaeva
- Subjects
0301 basic medicine ,Ciliopathies ,03 medical and health sciences ,Otolithic Membrane ,0302 clinical medicine ,medicine ,Animals ,Secretion ,Amino Acid Sequence ,Cilia ,Bardet-Biedl Syndrome ,Secretory pathway ,Zebrafish ,Vestibular system ,Extracellular Matrix Proteins ,Multidisciplinary ,Base Sequence ,Chemistry ,Cilium ,Homozygote ,Gene Expression Regulation, Developmental ,Epistasis, Genetic ,Biological Sciences ,Zebrafish Proteins ,medicine.disease ,Cell biology ,Transport protein ,Ciliopathy ,030104 developmental biology ,medicine.anatomical_structure ,Phenotype ,Mutation ,Basal lamina ,sense organs ,Crystallization ,030217 neurology & neurosurgery - Abstract
Here, we report that important regulators of cilia formation and ciliary compartment-directed protein transport function in secretion polarity. Mutations in cilia genes cep290 and bbs2, involved in human ciliopathies, affect apical secretion of Cochlin, a major otolith component and a determinant of calcium carbonate crystallization form. We show that Cochlin, defective in human auditory and vestibular disorder, DFNA9, is secreted from small specialized regions of vestibular system epithelia. Cells of these regions secrete Cochlin both apically into the ear lumen and basally into the basal lamina. Basally secreted Cochlin diffuses along the basal surface of vestibular epithelia, while apically secreted Cochlin is incorporated into the otolith. Mutations in a subset of ciliopathy genes lead to defects in Cochlin apical secretion, causing abnormal otolith crystallization and behavioral defects. This study reveals a class of ciliary proteins that are important for the polarity of secretion and delineate a secretory pathway that regulates biomineralization.
- Published
- 2021
15. Mapping the evolution of bornaviruses across geological timescales
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Robert J. Gifford
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Time Factors ,Range (biology) ,viruses ,Genome, Viral ,Biology ,Genome ,Microbiology ,DNA sequencing ,Virus ,03 medical and health sciences ,Genus ,Zoonoses ,Animals ,030304 developmental biology ,0303 health sciences ,Borna disease ,Multidisciplinary ,Base Sequence ,030306 microbiology ,Fossils ,Outbreak ,Geology ,Biological Sciences ,Biological Evolution ,Evolutionary biology ,Bornaviridae ,Commentary ,Identification (biology) - Abstract
It has always seemed likely that viruses originated early in the history of life. However, until the identification of endogenous viral elements (EVEs), there was little if any direct evidence for most virus groups ever having existed in the distant past (1). EVEs are virus-derived DNA sequences found in the germline genomes of metazoan species. Uniquely, they preserve information about the genomes of viruses that circulated tens to hundreds of millions of years ago. Comparative analysis of EVE sequences has now provided robust age calibrations for a diverse range of virus groups, completely transforming perspectives on the longer-term evolutionary interactions between viruses and hosts (2). As progress in mapping the complete genome sequences of species has accelerated, the abundance of “fossilized” viral sequences in eukaryotic genomes has become apparent. Increasingly, the challenge is not finding EVEs, but scaling analytical approaches to tackle the exponentially increasing volume of EVE sequence data (3, 4). In PNAS, Kawasaki et al. (5) utilize sophisticated computational approaches to implement a broad-scale analysis of the viral “fossil record,” focusing on a group of viruses called “bornaviruses.” Bornaviruses (family Bornaviridae ) are a poorly understood group of single-stranded negative sense RNA viruses (order Mononegavirales ) that infect vertebrates. Until 2015 the family contained only one genus ( Orthobornavirus ); however, recent progress in sampling viral diversity has led to the establishment of two novel genera ( Carbovirus and Curtervirus ) (6). The prototype member, Borna disease virus (BDV), infects a variety of mammalian species and is the causative agent of a neurological condition in horses referred to as Borna disease or “sad horse disease.” The name “Borna” derives from an 1895 outbreak that occurred in the vicinity of the town of Borna in Saxony, Germany, and decimated the Prussian cavalry (7). The association of BDV with neurological disorders in mammals, combined with … [↵][1]1Email: robert.gifford{at}glasgow.ac.uk. [1]: #xref-corresp-1-1
- Published
- 2021
16. Human cytomegalovirus haplotype reconstruction reveals high diversity due to superinfection and evidence of within-host recombination
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Cudini, Juliana, Roy, Sunando, Houldcroft, Charlotte J., Bryant, Josephine M., Depledge, Daniel P., Tutill, Helena, Veys, Paul, Williams, Rachel, Worth, Austen J. J., Tamuri, Asif U., Goldstein, Richard A., Breuer, Judith, Goldstein, Richard A [0000-0001-5148-4672], Breuer, Judith [0000-0001-8246-0534], and Apollo - University of Cambridge Repository
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Male ,viruses ,Cytomegalovirus ,Genome, Viral ,Microbiology ,diversity ,superinfection ,Immunocompromised Host ,Humans ,Child ,Recombination, Genetic ,whole genome sequencing ,Base Sequence ,Genome, Human ,Infant, Newborn ,Genetic Variation ,High-Throughput Nucleotide Sequencing ,Infant ,virus diseases ,Sequence Analysis, DNA ,biochemical phenomena, metabolism, and nutrition ,Biological Sciences ,recombination ,Haplotypes ,human cytomegalovirus ,Child, Preschool ,Cytomegalovirus Infections ,DNA, Viral ,Female - Abstract
Significance Human cytomegalovirus (HCMV) is a major global cause of congenital disability and transplant-related morbidity. Excessive levels of within-host HCMV nucleotide diversity are attributed to unexpectedly high mutation rates. Here, we show that high HCMV diversity is due to the frequent presence of mixed infections with genetically distinct strains, whereas HCMV in nonmixed infections is no more diverse than other DNA viruses. Using serial patient samples, we reconstruct viral strain haplotypes to pinpoint the timing of HCMV superinfections occurring within the study sampling time frame and uncover within-host viral recombination. From these results, we identify likely sources of infection and demonstrate probable selection for recombinant viruses. These results generate new, yet testable, insights into putative viral and host drivers of HCMV evolution and pathogenesis., Recent sequencing efforts have led to estimates of human cytomegalovirus (HCMV) genome-wide intrahost diversity that rival those of persistent RNA viruses [Renzette N, Bhattacharjee B, Jensen JD, Gibson L, Kowalik TF (2011) PLoS Pathog 7:e1001344]. Here, we deep sequence HCMV genomes recovered from single and longitudinally collected blood samples from immunocompromised children to show that the observations of high within-host HCMV nucleotide diversity are explained by the frequent occurrence of mixed infections caused by genetically distant strains. To confirm this finding, we reconstructed within-host viral haplotypes from short-read sequence data. We verify that within-host HCMV nucleotide diversity in unmixed infections is no greater than that of other DNA viruses analyzed by the same sequencing and bioinformatic methods and considerably less than that of human immunodeficiency and hepatitis C viruses. By resolving individual viral haplotypes within patients, we reconstruct the timing, likely origins, and natural history of superinfecting strains. We uncover evidence for within-host recombination between genetically distinct HCMV strains, observing the loss of the parental virus containing the nonrecombinant fragment. The data suggest selection for strains containing the recombinant fragment, generating testable hypotheses about HCMV evolution and pathogenesis. These results highlight that high HCMV diversity present in some samples is caused by coinfection with multiple distinct strains and provide reassurance that within the host diversity for single-strain HCMV infections is no greater than for other herpesviruses.
- Published
- 2019
17. Virus-like insertions with sequence signatures similar to those of endogenous nonretroviral RNA viruses in the human genome
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Keizo Tomonaga, Jumpei Ito, Masayuki Horie, Shohei Kojima, Kohei Yoshikawa, Shuichi Kawano, Nicholas F. Parrish, and So Nakagawa
- Subjects
Mammals ,Multidisciplinary ,biology ,Base Sequence ,Genome, Human ,viruses ,RNA ,RNA virus ,Biological Sciences ,biology.organism_classification ,Genome ,Virus ,Machine Learning ,Mutagenesis, Insertional ,Nucleoproteins ,Retroviridae ,Evolutionary biology ,Viral evolution ,Animals ,Humans ,Human genome ,Paleovirology ,Reference genome - Abstract
Understanding the genetics and taxonomy of ancient viruses will give us great insights into not only the origin and evolution of viruses but also how viral infections played roles in our evolution. Endogenous viruses are remnants of ancient viral infections and are thought to retain the genetic characteristics of viruses from ancient times. In this study, we used machine learning of endogenous RNA virus sequence signatures to identify viruses in the human genome that have not been detected or are already extinct. Here, we show that the k-mer occurrence of ancient RNA viral sequences remains similar to that of extant RNA viral sequences and can be differentiated from that of other human genome sequences. Furthermore, using this characteristic, we screened RNA viral insertions in the human reference genome and found virus-like insertions with phylogenetic and evolutionary features indicative of an exogenous origin but lacking homology to previously identified sequences. Our analysis indicates that animal genomes still contain unknown virus-derived sequences and provides a glimpse into the diversity of the ancient virosphere.
- Published
- 2021
18. Distinct RNA N-demethylation pathways catalyzed by nonheme iron ALKBH5 and FTO enzymes enable regulation of formaldehyde release rates
- Author
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Kevin J. Bruemmer, Eva J Ge, Christopher J. Chang, Joel D.W. Toh, Diana A. Iovan, Dan He, and Steven W. M. Crossley
- Subjects
0301 basic medicine ,Models, Molecular ,Oxygenase ,Protein Conformation ,Mutant ,01 natural sciences ,chemistry.chemical_compound ,Models ,Cellular localization ,chemistry.chemical_classification ,Multidisciplinary ,biology ,Fatty Acids ,AlkB Homolog 5, RNA Demethylase ,RNA Demethylase ,Biochemistry ,Physical Sciences ,MCF-7 Cells ,Single-Cell Analysis ,Oxidation-Reduction ,one-carbon cycle ,1.1 Normal biological development and functioning ,Iron ,AlkB ,Alpha-Ketoglutarate-Dependent Dioxygenase FTO ,Oxidative phosphorylation ,010402 general chemistry ,RNA epigenetics ,03 medical and health sciences ,Underpinning research ,Humans ,Epigenetics ,AlkB Homolog 5 ,Demethylation ,nonheme iron enzyme ,Base Sequence ,RNA ,Molecular ,m6A ,0104 chemical sciences ,030104 developmental biology ,Enzyme ,HEK293 Cells ,chemistry ,biology.protein ,formaldehyde ,Generic health relevance ,N6-Methyladenosine - Abstract
The AlkB family of non-heme-Fe(II)/2-oxoglutarate(2OG)-dependent oxygenases are essential regulators of RNA epigenetics by serving as erasers of one-carbon marks on RNA with release of formaldehyde (FA). Two major human AlkB family members, FTO and ALKBH5, both act as oxidative demethylases of N6 methyladenosine (m6A) but furnish different major products, N6 hydroxymethyladenosine (hm6A) and adenosine (A), respectively. Here we identify foundational mechanistic differences between FTO and ALKBH5 that promote these distinct biochemical outcomes. In contrast to FTO, which follows a traditional oxidative N-demethylation pathway to catalyze conversion of m6A to hm6A with subsequent slow release of A and FA, we find that ALKBH5 catalyzes a directm6A-to-A transformation with rapid FA release. We identify a catalytic R130/K132/Y139 triad within ALKBH5 that facilitates release of FA via an unprecedented covalent-based demethylation mechanism with direct detection of a covalent intermediate. Importantly, a K132Q mutant furnishes an ALKBH5 enzyme with an m6A demethylation profile that resembles that of FTO, establishing the importance of this residue in the proposed covalent mechanism. Finally, we show that ALKBH5 is an endogenous source of FA in the cell by activity-based sensing of FA fluxes perturbed via ALKBH5 knockdown. This work provides a fundamental biochemical rationale for non-redundant roles of these RNA demethylases beyond different substrate preferences and cellular localization, where m6A demethylation by ALKBH5 versus FTO results in release of FA, an endogenous one-carbon unit but potential genotoxin, at different rates in living systems.Significance StatementNon-heme iron enzymes FTO and ALKBH5 play central roles in epigenetic RNA regulation by catalyzing the oxidation of N6-methyladenosine (m6A) to produce N6-hydroxymethyladenosine (hm6A) and adenosine (A), respectively. Here, we provide a mechanistic rationale for these distinct biochemical outcomes by identifying that ALKBH5 performs m6A demethylation via an unprecedented covalent-based mechanism with concomitant and rapid release of A and formaldehyde (FA), whereas FTO liberates hm6A to release A and FA over longer timescales. This work reveals foundational biochemical differences between these closely related but non-redundant epigenetic enzymes and identifies ALKBH5 as an endogenous source of rapid formaldehyde generation in cells.
- Published
- 2020
19. An RNA polymerase ribozyme that synthesizes its own ancestor
- Author
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Maxim N. Shokhirev, David P. Horning, Gerald F. Joyce, and Katrina F. Tjhung
- Subjects
chemistry.chemical_classification ,DNA ligase ,Multidisciplinary ,biology ,Base Sequence ,Nucleotides ,Ribozyme ,RNA ,Computational biology ,DNA-Directed RNA Polymerases ,Biological Sciences ,Non-coding RNA ,Directed evolution ,Polymerization ,chemistry.chemical_compound ,chemistry ,RNA polymerase ,biology.protein ,Nucleic Acid Conformation ,RNA, Catalytic ,Directed Molecular Evolution ,Nucleoside ,Polymerase - Abstract
The RNA-based organisms from which modern life is thought to have descended would have depended on an RNA polymerase ribozyme to copy functional RNA molecules, including copying the polymerase itself. Such a polymerase must have been capable of copying structured RNAs with high efficiency and high fidelity to maintain genetic information across successive generations. Here the class I RNA polymerase ribozyme was evolved in vitro for the ability to synthesize functional ribozymes, resulting in the markedly improved ability to synthesize complex RNAs using nucleoside 5′-triphosphate (NTP) substrates. The polymerase is descended from the class I ligase, which contains the same catalytic core as the polymerase. The class I ligase can be synthesized by the improved polymerase as three separate RNA strands that assemble to form a functional ligase. The polymerase also can synthesize the complement of each of these three strands. Despite this remarkable level of activity, only a very small fraction of the assembled ligases retain catalytic activity due to the presence of disabling mutations. Thus, the fidelity of RNA polymerization should be considered a major impediment to the construction of a self-sustained, RNA-based evolving system. The propagation of heritable information requires both efficient and accurate synthesis of genetic molecules, a requirement relevant to both laboratory systems and the early history of life on Earth.
- Published
- 2020
20. Taxonomic classification of DNA sequences beyond sequence similarity using deep neural networks.
- Author
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Mock F, Kretschmer F, Kriese A, Böcker S, and Marz M
- Subjects
- Algorithms, Base Sequence, Genome, Genomics, DNA classification, DNA genetics, DNA Barcoding, Taxonomic methods, Deep Learning, Software
- Abstract
Taxonomic classification, that is, the assignment to biological clades with shared ancestry, is a common task in genetics, mainly based on a genome similarity search of large genome databases. The classification quality depends heavily on the database, since representative relatives must be present. Many genomic sequences cannot be classified at all or only with a high misclassification rate. Here we present BERTax, a deep neural network program based on natural language processing to precisely classify the superkingdom and phylum of DNA sequences taxonomically without the need for a known representative relative from a database. We show BERTax to be at least on par with the state-of-the-art approaches when taxonomically similar species are part of the training data. For novel organisms, however, BERTax clearly outperforms any existing approach. Finally, we show that BERTax can also be combined with database approaches to further increase the prediction quality in almost all cases. Since BERTax is not based on similar entries in databases, it allows precise taxonomic classification of a broader range of genomic sequences, thus increasing the overall information gain.
- Published
- 2022
- Full Text
- View/download PDF
21. Measuring thermodynamic preferences to form non-native conformations in nucleic acids using ultraviolet melting.
- Author
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Rangadurai A, Shi H, Xu Y, Liu B, Abou Assi H, Boom JD, Zhou H, Kimsey IJ, and Al-Hashimi HM
- Subjects
- Base Sequence, Freezing, Thermodynamics, Ultraviolet Rays, DNA chemistry, Nucleic Acid Conformation, RNA chemistry
- Abstract
Thermodynamic preferences to form non-native conformations are crucial for understanding how nucleic acids fold and function. However, they are difficult to measure experimentally because this requires accurately determining the population of minor low-abundance (<10%) conformations in a sea of other conformations. Here, we show that melting experiments enable facile measurements of thermodynamic preferences to adopt nonnative conformations in DNA and RNA. The key to this "delta-melt" approach is to use chemical modifications to render specific minor non-native conformations the major state. The validity and robustness of delta-melt is established for four different non-native conformations under various physiological conditions and sequence contexts through independent measurements of thermodynamic preferences using NMR. Delta-melt is faster relative to NMR, simple, and cost-effective and enables thermodynamic preferences to be measured for exceptionally low-populated conformations. Using delta-melt, we obtained rare insights into conformational cooperativity, obtaining evidence for significant cooperativity (1.0 to 2.5 kcal/mol) when simultaneously forming two adjacent Hoogsteen base pairs. We also measured the thermodynamic preferences to form G-C
+ and A-T Hoogsteen and A-T base open states for nearly all 16 trinucleotide sequence contexts and found distinct sequence-specific variations on the order of 2 to 3 kcal/mol. This rich landscape of sequence-specific non-native minor conformations in the DNA double helix may help shape the sequence specificity of DNA biochemistry. Thus, melting experiments can now be used to access thermodynamic information regarding regions of the free energy landscape of biomolecules beyond the native folded and unfolded conformations.- Published
- 2022
- Full Text
- View/download PDF
22. Retraction for Cifuentes-Rojas et al., Two RNA subunits and POT1a are components of Arabidopsis telomerase
- Subjects
Base Sequence ,Arabidopsis Proteins ,Reverse Transcriptase Polymerase Chain Reaction ,Blotting, Western ,Molecular Sequence Data ,Arabidopsis ,Oligonucleotides ,food and beverages ,Nuclear Proteins ,Sequence Analysis, DNA ,Biological Sciences ,Blotting, Northern ,Retraction ,Protein Subunits ,Gene Duplication ,Mutation ,Mutagenesis, Site-Directed ,Immunoprecipitation ,RNA ,Nucleic Acid Amplification Techniques ,Telomerase ,DNA Primers - Abstract
Telomerase is a ribonucleoprotein (RNP) reverse transcriptase whose essential RNA subunit (TER) functions as a template for telomere repeat synthesis. Here we report the identification of two divergent TER moieties in the flowering plant Arabidopsis thaliana. Although both TER1 and TER2 copurify with telomerase activity and serve as templates for telomerase in vitro, depletion of TER1, but not TER2, leads to decreased telomerase activity and progressive telomere shortening in vivo. Moreover, mutation of the templating domain in TER1 results in the incorporation of mutant telomere repeats on chromosome ends. Thus, TER1 provides the major template for telomerase in vivo. We also show that POT1a binds TER1 with a Kd of 2 × 10(-7) M and the two components assemble into an enzymatically active RNP in vivo. In contrast, TER1-POT1b and TER2-POT1a associations were not observed. In other organisms POT1 proteins bind telomeric DNA and provide chromosome end protection. We propose that duplication of TER and POT1 in Arabidopsis fueled the evolution of novel protein-nucleic acid interactions and the migration of POT1 from the telomere to the telomerase RNP.
- Published
- 2019
23. Comprehensive, high-resolution binding energy landscapes reveal context dependencies of transcription factor binding
- Author
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Polly M. Fordyce, Allison M. Keys, Yaron Orenstein, Daniel D. Le, Tyler C. Shimko, Scott A. Longwell, and Arjun K. Aditham
- Subjects
0301 basic medicine ,Saccharomyces cerevisiae Proteins ,Consensus site ,Transcription, Genetic ,Binding energy ,protein–DNA binding ,microfluidics ,Pho4 ,Computational biology ,Biology ,DNA sequencing ,E-Box Elements ,03 medical and health sciences ,chemistry.chemical_compound ,Transcription (biology) ,transcriptional regulation ,Computer Simulation ,Transcription factor ,Gene Library ,Genetics ,Multidisciplinary ,Binding Sites ,Base Sequence ,Basic Helix-Loop-Helix Leucine Zipper Transcription Factors ,High-Throughput Nucleotide Sequencing ,DNA ,Sequence Analysis, DNA ,Biological Sciences ,Microfluidic Analytical Techniques ,DNA binding site ,transcription factor specificity ,DNA-Binding Proteins ,Biophysics and Computational Biology ,030104 developmental biology ,transcription factor binding ,chemistry ,PNAS Plus ,Physical Sciences ,Thermodynamics ,Monte Carlo Method ,Protein Binding ,Transcription Factors - Abstract
Significance Transcription factors (TFs) are key proteins that bind DNA targets to coordinate gene expression in cells. Understanding how TFs recognize their DNA targets is essential for predicting how variations in regulatory sequence disrupt transcription to cause disease. Here, we develop a high-throughput assay and analysis pipeline capable of measuring binding energies for over one million sequences with high resolution and apply it toward understanding how nucleotides flanking DNA targets affect binding energies for two model yeast TFs. Through systematic comparisons between models trained on these data, we establish that considering dinucleotide (DN) interactions is sufficient to accurately predict binding and further show that sites used by TFs in vivo are both energetically and mutationally distant from the highest affinity sequence., Transcription factors (TFs) are primary regulators of gene expression in cells, where they bind specific genomic target sites to control transcription. Quantitative measurements of TF–DNA binding energies can improve the accuracy of predictions of TF occupancy and downstream gene expression in vivo and shed light on how transcriptional networks are rewired throughout evolution. Here, we present a sequencing-based TF binding assay and analysis pipeline (BET-seq, for Binding Energy Topography by sequencing) capable of providing quantitative estimates of binding energies for more than one million DNA sequences in parallel at high energetic resolution. Using this platform, we measured the binding energies associated with all possible combinations of 10 nucleotides flanking the known consensus DNA target interacting with two model yeast TFs, Pho4 and Cbf1. A large fraction of these flanking mutations change overall binding energies by an amount equal to or greater than consensus site mutations, suggesting that current definitions of TF binding sites may be too restrictive. By systematically comparing estimates of binding energies output by deep neural networks (NNs) and biophysical models trained on these data, we establish that dinucleotide (DN) specificities are sufficient to explain essentially all variance in observed binding behavior, with Cbf1 binding exhibiting significantly more nonadditivity than Pho4. NN-derived binding energies agree with orthogonal biochemical measurements and reveal that dynamically occupied sites in vivo are both energetically and mutationally distant from the highest affinity sites.
- Published
- 2018
24. RNAi is a critical determinant of centromere evolution in closely related fungi
- Author
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Terrance Shea, Christina A. Cuomo, Kaustuv Sanyal, Robert E. Lintner, R. Blake Billmyre, Joseph Heitman, Guus Bakkeren, Sheng Sun, Vikas Yadav, and Bhagya C. Thimmappa
- Subjects
0301 basic medicine ,Transposable element ,Centromere ,Locus (genetics) ,Retrotransposon ,Biology ,DNA sequencing ,Evolution, Molecular ,03 medical and health sciences ,chemistry.chemical_compound ,Genetics ,Ustilago ,experimental evolution ,DNA, Fungal ,Multidisciplinary ,DNA methylation ,Base Sequence ,Kinetochore ,fungi ,Biological Sciences ,retrotransposons ,Cryptococcus ,030104 developmental biology ,chemistry ,Eukaryotic chromosome fine structure ,RNA Interference ,Chromosomes, Fungal ,DNA - Abstract
Significance The “centromere paradox” refers to rapidly evolving and highly diverse centromere DNA sequences even in closely related eukaryotes. However, factors contributing to this rapid divergence are largely unknown. Here, we identified large regional, LTR retrotransposon-rich centromeres in a group of human fungal pathogens belonging to the Cryptococcus species complex. We provide evidence that loss-of-functional RNAi machinery and possibly cytosine DNA methylation trigger instability of the genome by activation of centromeric retrotransposons presumably suppressed by RNAi. We propose that RNAi, together with cytosine DNA methylation, serves as a critical determinant that maintains repetitive transposon-rich centromere structures. This study explores the direct link between RNAi and centromere structure evolution., The centromere DNA locus on a eukaryotic chromosome facilitates faithful chromosome segregation. Despite performing such a conserved function, centromere DNA sequence as well as the organization of sequence elements is rapidly evolving in all forms of eukaryotes. The driving force that facilitates centromere evolution remains an enigma. Here, we studied the evolution of centromeres in closely related species in the fungal phylum of Basidiomycota. Using ChIP-seq analysis of conserved inner kinetochore proteins, we identified centromeres in three closely related Cryptococcus species: two of which are RNAi-proficient, while the other lost functional RNAi. We find that the centromeres in the RNAi-deficient species are significantly shorter than those of the two RNAi-proficient species. While centromeres are LTR retrotransposon-rich in all cases, the RNAi-deficient species lost all full-length retroelements from its centromeres. In addition, centromeres in RNAi-proficient species are associated with a significantly higher level of cytosine DNA modifications compared with those of RNAi-deficient species. Furthermore, when an RNAi-proficient Cryptococcus species and its RNAi-deficient mutants were passaged under similar conditions, the centromere length was found to be occasionally shortened in RNAi mutants. In silico analysis of predicted centromeres in a group of closely related Ustilago species, also belonging to the Basidiomycota, were found to have undergone a similar transition in the centromere length in an RNAi-dependent fashion. Based on the correlation found in two independent basidiomycetous species complexes, we present evidence suggesting that the loss of RNAi and cytosine DNA methylation triggered transposon attrition, which resulted in shortening of centromere length during evolution.
- Published
- 2018
25. Triple-helix potential of the mouse genome.
- Author
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Maekawa K, Yamada S, Sharma R, Chaudhuri J, and Keeney S
- Subjects
- Animals, Base Sequence, Mice, Nucleic Acid Conformation, Genome genetics, Nucleotide Motifs
- Abstract
Certain DNA sequences, including mirror-symmetric polypyrimidine•polypurine runs, are capable of folding into a triple-helix–containing non–B-form DNA structure called H-DNA. Such H-DNA–forming sequences occur frequently in many eukaryotic genomes, including in mammals, and multiple lines of evidence indicate that these motifs are mutagenic and can impinge on DNA replication, transcription, and other aspects of genome function. In this study, we show that the triplex-forming potential of H-DNA motifs in the mouse genome can be evaluated using S1-sequencing (S1-seq), which uses the single-stranded DNA (ssDNA)–specific nuclease S1 to generate deep-sequencing libraries that report on the position of ssDNA throughout the genome. When S1-seq was applied to genomic DNA isolated from mouse testis cells and splenic B cells, we observed prominent clusters of S1-seq reads that appeared to be independent of endogenous double-strand breaks, that coincided with H-DNA motifs, and that correlated strongly with the triplex-forming potential of the motifs. Fine-scale patterns of S1-seq reads, including a pronounced strand asymmetry in favor of centrally positioned reads on the pyrimidine-containing strand, suggested that this S1-seq signal is specific for one of the four possible isomers of H-DNA (H-y5). By leveraging the abundance and complexity of naturally occurring H-DNA motifs across the mouse genome, we further defined how polypyrimidine repeat length and the presence of repeat-interrupting substitutions modify the structure of H-DNA. This study provides an approach for studying DNA secondary structure genome-wide at high spatial resolution.
- Published
- 2022
- Full Text
- View/download PDF
26. Species-specific KRAB-ZFPs function as repressors of retroviruses by targeting PBS regions.
- Author
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Yang B, Fang L, Gao Q, Xu C, Xu J, Chen ZX, Wang Y, and Yang P
- Subjects
- Base Sequence, Binding Sites, Chromosome Mapping, Endogenous Retroviruses, Genome-Wide Association Study, Humans, Nucleotide Motifs, Retroviridae classification, Transcription, Genetic, Virus Replication, Gene Expression Regulation, Viral, Host-Pathogen Interactions, RNA, Viral genetics, RNA, Viral metabolism, Repressor Proteins metabolism, Retroviridae physiology, Zinc Fingers
- Abstract
SignificanceHosts often target the relatively conserved regions in rapidly mutating retroviruses to inhibit their replication. One of these regions is called a primer binding site (PBS), which has to be complementary to the host tRNA to initiate reverse transcription. By analyzing endogenous retroviral elements, we found that host cells use this sequence as a target in efforts to block the expression of viral elements. A specific type of zinc finger protein targets the PBS in a host genome, which not only inhibits the transcription of endogenous viruses but also inhibits the replication of exogenous retroviruses with the same PBS. Thus, our study sheds light on a strategy for searching for host restriction factors targeting retroviruses.
- Published
- 2022
- Full Text
- View/download PDF
27. Targeting stem-loop 1 of the SARS-CoV-2 5' UTR to suppress viral translation and Nsp1 evasion.
- Author
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Vora SM, Fontana P, Mao T, Leger V, Zhang Y, Fu TM, Lieberman J, Gehrke L, Shi M, Wang L, Iwasaki A, and Wu H
- Subjects
- Animals, Base Sequence, Chlorocebus aethiops, HEK293 Cells, Host-Pathogen Interactions drug effects, Host-Pathogen Interactions genetics, Humans, Immune Evasion drug effects, Mice, Transgenic, Models, Biological, Oligonucleotides, Antisense pharmacology, SARS-CoV-2 drug effects, Vero Cells, Virus Replication drug effects, 5' Untranslated Regions genetics, Immune Evasion genetics, Protein Biosynthesis drug effects, SARS-CoV-2 genetics, Viral Nonstructural Proteins genetics
- Abstract
SARS-CoV-2 is a highly pathogenic virus that evades antiviral immunity by interfering with host protein synthesis, mRNA stability, and protein trafficking. The SARS-CoV-2 nonstructural protein 1 (Nsp1) uses its C-terminal domain to block the messenger RNA (mRNA) entry channel of the 40S ribosome to inhibit host protein synthesis. However, how SARS-CoV-2 circumvents Nsp1-mediated suppression for viral protein synthesis and if the mechanism can be targeted therapeutically remain unclear. Here, we show that N- and C-terminal domains of Nsp1 coordinate to drive a tuned ratio of viral to host translation, likely to maintain a certain level of host fitness while maximizing replication. We reveal that the stem-loop 1 (SL1) region of the SARS-CoV-2 5' untranslated region (5' UTR) is necessary and sufficient to evade Nsp1-mediated translational suppression. Targeting SL1 with locked nucleic acid antisense oligonucleotides inhibits viral translation and makes SARS-CoV-2 5' UTR vulnerable to Nsp1 suppression, hindering viral replication in vitro at a nanomolar concentration, as well as providing protection against SARS-CoV-2-induced lethality in transgenic mice expressing human ACE2. Thus, SL1 allows Nsp1 to switch infected cells from host to SARS-CoV-2 translation, presenting a therapeutic target against COVID-19 that is conserved among immune-evasive variants. This unique strategy of unleashing a virus' own virulence mechanism against itself could force a critical trade-off between drug resistance and pathogenicity., Competing Interests: The authors declare no competing interest., (Copyright © 2022 the Author(s). Published by PNAS.)
- Published
- 2022
- Full Text
- View/download PDF
28. Sequence-dependent RNA helix conformational preferences predictably impact tertiary structure formation
- Author
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Joseph D. Yesselman, Rhiju Das, William J. Greenleaf, Namita Bisaria, Sarah K. Denny, and Daniel Herschlag
- Subjects
Models, Molecular ,Multidisciplinary ,Base Sequence ,Nucleic acid tertiary structure ,Chemistry ,Base pair ,RNA Stability ,RNA ,Sequence (biology) ,Protein tertiary structure ,PNAS Plus ,Helix ,Gene expression ,Biophysics ,Nucleic Acid Conformation ,Thermodynamics ,Nucleic acid structure ,Base Pairing - Abstract
Structured RNAs and RNA complexes underlie biological processes ranging from control of gene expression to protein translation. Approximately 50% of nucleotides within known structured RNAs are folded into Watson-Crick (WC) base pairs, and sequence changes that preserve these pairs are typically assumed to preserve higher-order RNA structure and binding of macromolecule partners. Here, we report that indirect effects of the helix sequence on RNA tertiary stability are, in fact, significant but are nevertheless predictable from a simple computational model called RNAMake-∆∆G. When tested through the RNA on a massively parallel array (RNA-MaP) experimental platform, blind predictions for >1500 variants of the tectoRNA heterodimer model system achieve high accuracy (rmsd 0.34 and 0.77 kcal/mol for sequence and length changes, respectively). Detailed comparison of predictions to experiments support a microscopic picture of how helix sequence changes subtly modulate conformational fluctuations at each base-pair step, which accumulate to impact RNA tertiary structure stability. Our study reveals a previously overlooked phenomenon in RNA structure formation and provides a framework of computation and experiment for understanding helix conformational preferences and their impact across biological RNA and RNA-protein assemblies.
- Published
- 2019
29. Ebolavirus polymerase uses an unconventional genome replication mechanism
- Author
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Rachel Fearns, Tessa N. Cressey, Laure R. Deflubé, Friederike Feldmann, Elke Mühlberger, Judith Olejnik, Hideki Ebihara, Adam J. Hume, and Elaine Haddock
- Subjects
viruses ,Genome, Viral ,Biology ,medicine.disease_cause ,Virus Replication ,Genome ,Article ,medicine ,Humans ,Nucleotide ,Polymerase ,Immune Evasion ,Ebolavirus ,Genetics ,chemistry.chemical_classification ,Multidisciplinary ,Base Sequence ,Nucleotides ,RNA ,RNA virus ,Hemorrhagic Fever, Ebola ,biology.organism_classification ,chemistry ,Viral replication ,PNAS Plus ,Replication Initiation ,biology.protein ,RNA, Viral - Abstract
Most nonsegmented negative strand (NNS) RNA virus genomes have complementary 3' and 5' terminal nucleotides because the promoters at the 3' ends of the genomes and antigenomes are almost identical to each other. However, according to published sequences, both ends of ebolavirus genomes show a high degree of variability, and the 3' and 5' terminal nucleotides are not complementary. If correct, this would distinguish the ebolaviruses from other NNS RNA viruses. Therefore, we investigated the terminal genomic and antigenomic nucleotides of three different ebolavirus species, Ebola (EBOV), Sudan, and Reston viruses. Whereas the 5' ends of ebolavirus RNAs are highly conserved with the sequence ACAGG-5', the 3' termini are variable and are typically 3'-GCCUGU, ACCUGU, or CCUGU. A small fraction of analyzed RNAs had extended 3' ends. The majority of 3' terminal sequences are consistent with a mechanism of nucleotide addition by hairpin formation and back-priming. Using single-round replicating EBOV minigenomes, we investigated the effect of the 3' terminal nucleotide on viral replication and found that the EBOV polymerase initiates replication opposite the 3'-CCUGU motif regardless of the identity of the 3' terminal nucleotide(s) and of the position of this motif relative to the 3' end. Deletion or mutation of the first residue of the 3'-CCUGU motif completely abolished replication initiation, suggesting a crucial role of this nucleotide in directing initiation. Together, our data show that ebolaviruses have evolved a unique replication strategy among NNS RNA viruses resulting in 3' overhangs. This could be a mechanism to avoid antiviral recognition.
- Published
- 2019
30. Evolutionarily informed deep learning methods for predicting relative transcript abundance from DNA sequence
- Author
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Jacob D. Washburn, Guillaume P. Ramstein, Edward S. Buckler, Maria Katherine Mejia Guerra, Karl A. Kremling, Ravi Valluru, and Hai Wang
- Subjects
Untranslated region ,Multidisciplinary ,Base Sequence ,Transcription, Genetic ,business.industry ,Deep learning ,D400 Agriculture ,Context (language use) ,Computational biology ,DNA ,Sequence Analysis, DNA ,Biology ,Models, Theoretical ,DNA sequencing ,Evolution, Molecular ,Deep Learning ,PNAS Plus ,Gene Expression Regulation ,False positive paradox ,Leverage (statistics) ,Gene family ,Artificial intelligence ,business ,Spurious relationship - Abstract
Deep learning methodologies have revolutionized prediction in many fields and show potential to do the same in molecular biology and genetics. However, applying these methods in their current forms ignores evolutionary dependencies within biological systems and can result in false positives and spurious conclusions. We developed two approaches that account for evolutionary relatedness in machine learning models: ( i ) gene-family–guided splitting and ( ii ) ortholog contrasts. The first approach accounts for evolution by constraining model training and testing sets to include different gene families. The second approach uses evolutionarily informed comparisons between orthologous genes to both control for and leverage evolutionary divergence during the training process. The two approaches were explored and validated within the context of mRNA expression level prediction and have the area under the ROC curve (auROC) values ranging from 0.75 to 0.94. Model weight inspections showed biologically interpretable patterns, resulting in the hypothesis that the 3′ UTR is more important for fine-tuning mRNA abundance levels while the 5′ UTR is more important for large-scale changes.
- Published
- 2019
31. Correction for Narayanan et al., Prefrontal D1 dopamine signaling is required for temporal control
- Author
-
Nandakumar Narayanan
- Subjects
Male ,Multidisciplinary ,Time Factors ,Base Sequence ,Behavior, Animal ,Tyrosine 3-Monooxygenase ,Receptors, Dopamine D1 ,Models, Neurological ,Ventral Tegmental Area ,Prefrontal Cortex ,Mice, Transgenic ,Biological Sciences ,Corrections ,Synaptic Transmission ,Rats ,Optogenetics ,Mice ,Reward ,Biological Clocks ,Neural Pathways ,Animals ,RNA Interference ,RNA, Small Interfering ,Signal Transduction - Abstract
Temporal control, or how organisms guide movements in time to achieve behavioral goals, depends on dopamine signaling. The medial prefrontal cortex controls many goal-directed behaviors and receives dopaminergic input primarily from the midbrain ventral tegmental area. However, this system has never been linked with temporal control. Here, we test the hypothesis that dopaminergic projections from the ventral tegmental area to the prefrontal cortex influence temporal control. Rodents were trained to perform a fixed-interval timing task with an interval of 20 s. We report several results: first, that decreasing dopaminergic neurotransmission using virally mediated RNA interference of tyrosine hydroxylase impaired temporal control, and second that pharmacological disruption of prefrontal D1 dopamine receptors, but not D2 dopamine receptors, impaired temporal control. We then used optogenetics to specifically and selectively manipulate prefrontal neurons expressing D1 dopamine receptors during fixed-interval timing performance. Selective inhibition of D1-expressing prefrontal neurons impaired fixed-interval timing, whereas stimulation made animals more efficient during task performance. These data provide evidence that ventral tegmental dopaminergic projections to the prefrontal cortex influence temporal control via D1 receptors. The results identify a critical circuit for temporal control of behavior that could serve as a target for the treatment of dopaminergic diseases.
- Published
- 2019
32. Correction for Liu et al., The genome landscape of ERα- and ERβ-binding DNA regions
- Author
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Jan-Ake Gustafsson, Hui Gao, Yawen Liu, Karin Dahlman-Wright, Anders Ström, Albin Sandelin, Eivind Valen, and Troels Marstrand
- Subjects
Genetics ,Chromatin Immunoprecipitation ,Multidisciplinary ,Binding Sites ,Genome ,Base Sequence ,Estrogen Receptor alpha ,DNA ,Biology ,Corrections ,Antibodies ,chemistry.chemical_compound ,chemistry ,Cell Line, Tumor ,Estrogen Receptor beta ,Humans ,Protein Binding - Abstract
In this article, we have applied the ChIP-on-chip approach to pursue a large scale identification of ERalpha- and ERbeta-binding DNA regions in intact chromatin. We show that there is a high degree of overlap between the regions identified as bound by ERalpha and ERbeta, respectively, but there are also regions that are bound by ERalpha only in the presence of ERbeta, as well as regions that are selectively bound by either receptor. Analysis of bound regions shows that regions bound by ERalpha have distinct properties in terms of genome landscape, sequence features, and conservation compared with regions that are bound by ERbeta. ERbeta-bound regions are, as a group, located more closely to transcription start sites. ERalpha- and ERbeta-bound regions differ in sequence properties, with ERalpha-bound regions having an overrepresentation of TA-rich motifs including forkhead binding sites and ERbeta-bound regions having a predominance of classical estrogen response elements (EREs) and GC-rich motifs. Differences in the properties of ER bound regions might explain some of the differences in gene expression programs and physiological effects shown by the respective estrogen receptors.
- Published
- 2019
33. Nonenzymatic assembly of active chimeric ribozymes from aminoacylated RNA oligonucleotides.
- Author
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Radakovic A, DasGupta S, Wright TH, Aitken HRM, and Szostak JW
- Subjects
- Base Sequence, DNA, Nucleic Acid Conformation, RNA, Transfer genetics, RNA, Transfer metabolism, RNA, Catalytic metabolism, Transfer RNA Aminoacylation physiology
- Abstract
Aminoacylated transfer RNAs, which harbor a covalent linkage between amino acids and RNA, are a universally conserved feature of life. Because they are essential substrates for ribosomal translation, aminoacylated oligonucleotides must have been present in the RNA world prior to the evolution of the ribosome. One possibility we are exploring is that the aminoacyl ester linkage served another function before being recruited for ribosomal protein synthesis. The nonenzymatic assembly of ribozymes from short RNA oligomers under realistic conditions remains a key challenge in demonstrating a plausible pathway from prebiotic chemistry to the RNA world. Here, we show that aminoacylated RNAs can undergo template-directed assembly into chimeric amino acid-RNA polymers that are active ribozymes. We demonstrate that such chimeric polymers can retain the enzymatic function of their all-RNA counterparts by generating chimeric hammerhead, RNA ligase, and aminoacyl transferase ribozymes. Amino acids with diverse side chains form linkages that are well tolerated within the RNA backbone and, in the case of an aminoacyl transferase, even in its catalytic center, potentially bringing novel functionalities to ribozyme catalysis. Our work suggests that aminoacylation chemistry may have played a role in primordial ribozyme assembly. Increasing the efficiency of this process provides an evolutionary rationale for the emergence of sequence and amino acid-specific aminoacyl-RNA synthetase ribozymes, which could then have generated the substrates for ribosomal protein synthesis., Competing Interests: The authors declare no competing interest., (Copyright © 2022 the Author(s). Published by PNAS.)
- Published
- 2022
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34. Template switching in DNA replication can create and maintain RNA hairpins.
- Author
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Mönttinen HAM and Löytynoja A
- Subjects
- Base Pairing, Base Sequence, Mutation, RNA genetics, DNA chemistry, DNA genetics, DNA Replication, Inverted Repeat Sequences, Nucleic Acid Conformation, RNA chemistry, Templates, Genetic
- Abstract
The evolutionary origin of RNA stem structures and the preservation of their base pairing under a spontaneous and random mutation process have puzzled theoretical evolutionary biologists. DNA replication-related template switching is a mutation mechanism that creates reverse-complement copies of sequence regions within a genome by replicating briefly along either the complementary or nascent DNA strand. Depending on the relative positions and context of the four switch points, this process may produce a reverse-complement repeat capable of forming the stem of a perfect DNA hairpin or fix the base pairing of an existing stem. Template switching is typically thought to trigger large structural changes, and its possible role in the origin and evolution of RNA genes has not been studied. Here, we show that the reconstructed ancestral histories of RNA genes contain mutation patterns consistent with the DNA replication-related template switching. In addition to multibase compensatory mutations, the mechanism can explain complex sequence changes, although mutations breaking the structure rarely get fixed in evolution. Our results suggest a solution for the long-standing dilemma of RNA gene evolution and demonstrate how template switching can both create perfect stems with a single mutation event and help maintaining the stem structure over time. Interestingly, template switching also provides an elegant explanation for the asymmetric base pair frequencies within RNA stems., Competing Interests: The authors declare no competing interest., (Copyright © 2022 the Author(s). Published by PNAS.)
- Published
- 2022
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35. Recurrent mutations in topoisomerase IIα cause a previously undescribed mutator phenotype in human cancers.
- Author
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Boot A, Liu M, Stantial N, Shah V, Yu W, Nitiss KC, Nitiss JL, Jinks-Robertson S, and Rozen SG
- Subjects
- Alleles, Amino Acid Substitution, Base Sequence, Cell Survival, DNA Damage, DNA Mutational Analysis, DNA Topoisomerases, Type II metabolism, Gene Duplication, Gene Rearrangement, Genetic Predisposition to Disease, Genotype, Humans, INDEL Mutation, Mutagenesis, Neoplasms metabolism, Oncogenes, Poly-ADP-Ribose Binding Proteins genetics, Sequence Deletion, DNA Topoisomerases, Type II genetics, Mutation, Neoplasms genetics, Neoplasms pathology, Phenotype
- Abstract
Topoisomerases nick and reseal DNA to relieve torsional stress associated with transcription and replication and to resolve structures such as knots and catenanes. Stabilization of the yeast Top2 cleavage intermediates is mutagenic in yeast, but whether this extends to higher eukaryotes is less clear. Chemotherapeutic topoisomerase poisons also elevate cleavage, resulting in mutagenesis. Here, we describe p.K743N mutations in human topoisomerase hTOP2α and link them to a previously undescribed mutator phenotype in cancer. Overexpression of the orthologous mutant protein in yeast generated a characteristic pattern of 2- to 4-base pair (bp) duplications resembling those in tumors with p.K743N. Using mutant strains and biochemical analysis, we determined the genetic requirements of this mutagenic process and showed that it results from trapping of the mutant yeast yTop2 cleavage complex. In addition to 2- to 4-bp duplications, hTOP2α p.K743N is also associated with deletions that are absent in yeast. We call the combined pattern of duplications and deletions ID_TOP2α. All seven tumors carrying the hTOP2α p.K743N mutation showed ID_TOP2α, while it was absent from all other tumors examined ( n = 12,269). Each tumor with the ID_TOP2α signature had indels in several known cancer genes, which included frameshift mutations in tumor suppressors PTEN and TP53 and an activating insertion in BRAF. Sequence motifs found at ID_TOP2α mutations were present at 80% of indels in cancer-driver genes, suggesting that ID_TOP2α mutagenesis may contribute to tumorigenesis. The results reported here shed further light on the role of topoisomerase II in genome instability., Competing Interests: Competing interest statement: S.G.R., A.B., and one of the reviewers of this manuscript, Professor E.R., participated in the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which comprised over 700 researchers from around the world. S.G.R., A.B., and Professor E.R. did not directly work or publish together., (Copyright © 2022 the Author(s). Published by PNAS.)
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- 2022
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36. Reciprocal modulation of long noncoding RNA EMS and p53 regulates tumorigenesis.
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Wang C, Yang Y, Wu X, Li J, Liu K, Fang D, Li B, Shan G, Mei X, Wang F, and Mei Y
- Subjects
- Animals, Base Sequence, Cell Line, Tumor, Cell Proliferation genetics, Cellular Senescence genetics, Gene Expression Regulation, Neoplastic, HEK293 Cells, Humans, Male, Mice, Nude, Protein Biosynthesis, RNA, Long Noncoding genetics, RNA-Binding Proteins genetics, RNA-Binding Proteins metabolism, Transcription, Genetic, Mice, Carcinogenesis genetics, RNA, Long Noncoding metabolism, Tumor Suppressor Protein p53 metabolism
- Abstract
p53 plays a central role in tumor suppression. Emerging evidence suggests long noncoding RNA (lncRNA) as an important class of regulatory molecules that control the p53 signaling. Here, we report that the oncogenic lncRNA E2F1 messenger RNA (mRNA) stabilizing factor (EMS) and p53 mutually repress each other's expression. EMS is negatively regulated by p53. As a direct transcriptional repression target of p53, EMS is surprisingly shown to inhibit p53 expression. EMS associates with cytoplasmic polyadenylation element-binding protein 2 (CPEB2) and thus, disrupts the CPEB2-p53 mRNA interaction. This disassociation attenuates CPEB2-mediated p53 mRNA polyadenylation and suppresses p53 translation. Functionally, EMS is able to exert its oncogenic activities, at least partially, via the CPEB2-p53 axis. Together, these findings reveal a double-negative feedback loop between p53 and EMS, through which p53 is finely controlled. Our study also demonstrates a critical role for EMS in promoting tumorigenesis via the negative regulation of p53., Competing Interests: The authors declare no competing interest., (Copyright © 2022 the Author(s). Published by PNAS.)
- Published
- 2022
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37. Role of helical structure and dynamics in oligoadenylate synthetase 1 (OAS1) mismatch tolerance and activation by short dsRNAs.
- Author
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Schwartz SL, Dey D, Tanquary J, Bair CR, Lowen AC, and Conn GL
- Subjects
- Base Sequence, Endoribonucleases metabolism, Enzyme Activation, Humans, Inosine metabolism, Molecular Dynamics Simulation, Protein Structure, Secondary, RNA Editing, RNA Stability, Structure-Activity Relationship, Temperature, 2',5'-Oligoadenylate Synthetase chemistry, 2',5'-Oligoadenylate Synthetase metabolism, Base Pair Mismatch, RNA, Double-Stranded metabolism
- Abstract
The 2'-5'-oligoadenylate synthetases (OAS) are innate immune sensors of cytosolic double-stranded RNA (dsRNA) that play a critical role in limiting viral infection. How these proteins are able to avoid aberrant activation by cellular RNAs is not fully understood, but adenosine-to-inosine (A-to-I) editing has been proposed to limit accumulation of endogenous RNAs that might otherwise cause stimulation of the OAS/RNase L pathway. Here, we aim to uncover whether and how such sequence modifications can restrict the ability of short, defined dsRNAs to activate the single-domain form of OAS, OAS1. Unexpectedly, we find that all tested inosine-containing dsRNAs have an increased capacity to activate OAS1, whether in a destabilizing (I•U) or standard Watson-Crick-like base pairing (I-C) context. Additional variants with strongly destabilizing A•C mismatches or stabilizing G-C pairs also exhibit increased capacity to activate OAS1, eliminating helical stability as a factor in the relative ability of the dsRNAs to activate OAS1. Using thermal difference spectra and molecular dynamics simulations, we identify both increased helical dynamics and specific local changes in helical structure as important factors in the capacity of short dsRNAs to activate OAS1. These helical features may facilitate more ready adoption of the distorted OAS1-bound conformation or stabilize important structures to predispose the dsRNA for optimal binding and activation of OAS1. These studies thus reveal the molecular basis for the greater capacity of some short dsRNAs to activate OAS1 in a sequence-independent manner., Competing Interests: The authors declare no competing interest., (Copyright © 2022 the Author(s). Published by PNAS.)
- Published
- 2022
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38. The ectomycorrhizal fungus Pisolithus microcarpus encodes a microRNA involved in cross-kingdom gene silencing during symbiosis.
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Wong-Bajracharya J, Singan VR, Monti R, Plett KL, Ng V, Grigoriev IV, Martin FM, Anderson IC, and Plett JM
- Subjects
- Base Sequence, Basidiomycota growth & development, Colony Count, Microbial, Gene Expression Profiling, Gene Expression Regulation, Fungal, Genome, Fungal, MicroRNAs genetics, Plant Roots microbiology, RNA, Messenger genetics, RNA, Messenger metabolism, Basidiomycota genetics, Gene Silencing, MicroRNAs metabolism, Mycorrhizae genetics, Symbiosis genetics
- Abstract
Small RNAs (sRNAs) are known to regulate pathogenic plant-microbe interactions. Emerging evidence from the study of these model systems suggests that microRNAs (miRNAs) can be translocated between microbes and plants to facilitate symbiosis. The roles of sRNAs in mutualistic mycorrhizal fungal interactions, however, are largely unknown. In this study, we characterized miRNAs encoded by the ectomycorrhizal fungus Pisolithus microcarpus and investigated their expression during mutualistic interaction with Eucalyptus grandis. Using sRNA sequencing data and in situ miRNA detection, a novel fungal miRNA, Pmic_miR-8 , was found to be transported into E. grandis roots after interaction with P. microcarpus Further characterization experiments demonstrate that inhibition of Pmic_miR-8 negatively impacts the maintenance of mycorrhizal roots in E. grandis , while supplementation of Pmic_miR-8 led to deeper integration of the fungus into plant tissues. Target prediction and experimental testing suggest that Pmic_miR-8 may target the host NB-ARC domain containing transcripts, suggesting a potential role for this miRNA in subverting host signaling to stabilize the symbiotic interaction. Altogether, we provide evidence of previously undescribed cross-kingdom sRNA transfer from ectomycorrhizal fungi to plant roots, shedding light onto the involvement of miRNAs during the developmental process of mutualistic symbioses., Competing Interests: The authors declare no competing interest., (Copyright © 2022 the Author(s). Published by PNAS.)
- Published
- 2022
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39. Open reading frame correction using splice-switching antisense oligonucleotides for the treatment of cystic fibrosis.
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Michaels WE, Pena-Rasgado C, Kotaria R, Bridges RJ, and Hastings ML
- Subjects
- Alleles, Base Sequence, Bronchi pathology, Cell Line, Chloride Channels metabolism, Cystic Fibrosis pathology, Cystic Fibrosis Transmembrane Conductance Regulator genetics, Cystic Fibrosis Transmembrane Conductance Regulator metabolism, Epithelial Cells metabolism, Exons genetics, Homozygote, Humans, RNA, Messenger genetics, RNA, Messenger metabolism, Cystic Fibrosis genetics, Cystic Fibrosis therapy, Oligonucleotides, Antisense therapeutic use, Open Reading Frames genetics, RNA Splicing genetics
- Abstract
CFTR gene mutations that result in the introduction of premature termination codons (PTCs) are common in cystic fibrosis (CF). This mutation type causes a severe form of the disease, likely because of low CFTR messenger RNA (mRNA) expression as a result of nonsense-mediated mRNA decay, as well as the production of a nonfunctional, truncated CFTR protein. Current therapeutics for CF, which target residual protein function, are less effective in patients with these types of mutations due in part to low CFTR protein levels. Splice-switching antisense oligonucleotides (ASOs), designed to induce skipping of exons in order to restore the mRNA open reading frame, have shown therapeutic promise preclinically and clinically for a number of diseases. We hypothesized that ASO-mediated skipping of CFTR exon 23 would recover CFTR activity associated with terminating mutations in the exon, including CFTR p.W1282X, the fifth most common mutation in CF. Here, we show that CFTR lacking the amino acids encoding exon 23 is partially functional and responsive to corrector and modulator drugs currently in clinical use. ASO-induced exon 23 skipping rescued CFTR expression and chloride current in primary human bronchial epithelial cells isolated from a homozygote CFTR-W1282X patient. These results support the use of ASOs in treating CF patients with CFTR class I mutations in exon 23 that result in unstable CFTR mRNA and truncations of the CFTR protein., Competing Interests: Competing interest statement: M.L.H. is an inventor on issued patents US20160244767A1, US20180117073A1, and US20180119152A1—antisense compounds targeting genes associated with cystic fibrosis—assigned to Rosalind Franklin University of Medicine and Science., (Copyright © 2022 the Author(s). Published by PNAS.)
- Published
- 2022
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40. RNA polymerase II trapped on a molecular treadmill: Structural basis of persistent transcriptional arrest by a minor groove DNA binder.
- Author
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Oh J, Jia T, Xu J, Chong J, Dervan PB, and Wang D
- Subjects
- Base Sequence, Imidazoles chemistry, Models, Molecular, Pyrroles chemistry, Transcriptional Elongation Factors metabolism, DNA chemistry, Nucleic Acid Conformation, RNA Polymerase II metabolism, Transcription, Genetic
- Abstract
Elongating RNA polymerase II (Pol II) can be paused or arrested by a variety of obstacles. These obstacles include DNA lesions, DNA-binding proteins, and small molecules. Hairpin pyrrole-imidazole (Py-Im) polyamides bind to the minor groove of DNA in a sequence-specific manner and induce strong transcriptional arrest. Remarkably, this Py-Im-induced Pol II transcriptional arrest is persistent and cannot be rescued by transcription factor TFIIS. In contrast, TFIIS can effectively rescue the transcriptional arrest induced by a nucleosome barrier. The structural basis of Py-Im-induced transcriptional arrest and why TFIIS cannot rescue this arrest remain elusive. Here we determined the X-ray crystal structures of four distinct Pol II elongation complexes (Pol II ECs) in complex with hairpin Py-Im polyamides as well as of the hairpin Py-Im polyamides-dsDNA complex. We observed that the Py-Im oligomer directly interacts with RNA Pol II residues, introduces compression of the downstream DNA duplex, prevents Pol II forward translocation, and induces Pol II backtracking. These results, together with biochemical studies, provide structural insight into the molecular mechanism by which Py-Im blocks transcription. Our structural study reveals why TFIIS fails to promote Pol II bypass of Py-Im-induced transcriptional arrest., Competing Interests: The authors declare no competing interest., (Copyright © 2022 the Author(s). Published by PNAS.)
- Published
- 2022
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41. Mutations in artificial self-replicating tiles: A step toward Darwinian evolution.
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Zhou F, Sha R, Ni H, Seeman N, and Chaikin P
- Subjects
- Animals, Base Sequence, Biochemical Phenomena, DNA genetics, Mutation, Biological Evolution, DNA chemistry, DNA Replication physiology, Genetic Fitness
- Abstract
Artificial self-replication and exponential growth holds the promise of gaining a better understanding of fundamental processes in nature but also of evolving new materials and devices with useful properties. A system of DNA origami dimers has been shown to exhibit exponential growth and selection. Here we introduce mutation and growth advantages to study the possibility of Darwinian-like evolution. We seed and grow one dimer species, AB, from A and B monomers that doubles in each cycle. A similar species from C and D monomers can replicate at a controlled growth rate of two or four per cycle but is unseeded. Introducing a small mutation rate so that AB parents infrequently template CD offspring we show experimentally that the CD species can take over the system in approximately six generations in an advantageous environment. This demonstration opens the door to the use of evolution in materials design., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)
- Published
- 2021
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42. Observing the base-by-base search for native structure along transition paths during the folding of single nucleic acid hairpins.
- Author
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Hoffer NQ, Neupane K, and Woodside MT
- Subjects
- Base Pairing, Base Sequence, Thermodynamics, DNA chemistry, Nucleic Acid Conformation
- Abstract
Biomolecular folding involves searching among myriad possibilities for the native conformation, but the elementary steps expected from theory for this search have never been detected directly. We probed the dynamics of folding at high resolution using optical tweezers, measuring individual trajectories as nucleic acid hairpins passed through the high-energy transition states that dominate kinetics and define folding mechanisms. We observed brief but ubiquitous pauses in the transition states, with a dwell time distribution that matched microscopic theories of folding quantitatively. The sequence dependence suggested that pauses were dominated by microbarriers from nonnative conformations during the search by each nucleotide residue for the native base-pairing conformation. Furthermore, the pauses were position dependent, revealing subtle local variations in energy-landscape roughness and allowing the diffusion coefficient describing the microscopic dynamics within the barrier to be found without reconstructing the shape of the energy landscape. These results show how high-resolution measurements can elucidate key microscopic events during folding to test fundamental theories of folding., Competing Interests: The authors declare no competing interest.
- Published
- 2021
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43. Structure-function analysis of the nsp14 N7-guanine methyltransferase reveals an essential role in Betacoronavirus replication.
- Author
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Ogando NS, El Kazzi P, Zevenhoven-Dobbe JC, Bontes BW, Decombe A, Posthuma CC, Thiel V, Canard B, Ferron F, Decroly E, and Snijder EJ
- Subjects
- Amino Acid Sequence, Base Sequence, Binding Sites, Catalytic Domain, Conserved Sequence, Exoribonucleases genetics, Exoribonucleases metabolism, Microbial Viability, Nucleotide Motifs, RNA, Viral chemistry, RNA, Viral genetics, RNA-Binding Proteins, Structure-Activity Relationship, Viral Nonstructural Proteins genetics, Viral Nonstructural Proteins metabolism, Virus Replication genetics, Exoribonucleases chemistry, Models, Molecular, Protein Conformation, Viral Nonstructural Proteins chemistry
- Abstract
As coronaviruses (CoVs) replicate in the host cell cytoplasm, they rely on their own capping machinery to ensure the efficient translation of their messenger RNAs (mRNAs), protect them from degradation by cellular 5' exoribonucleases (ExoNs), and escape innate immune sensing. The CoV nonstructural protein 14 (nsp14) is a bifunctional replicase subunit harboring an N-terminal 3'-to-5' ExoN domain and a C-terminal (N7-guanine)-methyltransferase (N7-MTase) domain that is presumably involved in viral mRNA capping. Here, we aimed to integrate structural, biochemical, and virological data to assess the importance of conserved N7-MTase residues for nsp14's enzymatic activities and virus viability. We revisited the crystal structure of severe acute respiratory syndrome (SARS)-CoV nsp14 to perform an in silico comparative analysis between betacoronaviruses. We identified several residues likely involved in the formation of the N7-MTase catalytic pocket, which presents a fold distinct from the Rossmann fold observed in most known MTases. Next, for SARS-CoV and Middle East respiratory syndrome CoV, site-directed mutagenesis of selected residues was used to assess their importance for in vitro enzymatic activity. Most of the engineered mutations abolished N7-MTase activity, while not affecting nsp14-ExoN activity. Upon reverse engineering of these mutations into different betacoronavirus genomes, we identified two substitutions (R310A and F426A in SARS-CoV nsp14) abrogating virus viability and one mutation (H424A) yielding a crippled phenotype across all viruses tested. Our results identify the N7-MTase as a critical enzyme for betacoronavirus replication and define key residues of its catalytic pocket that can be targeted to design inhibitors with a potential pan-coronaviral activity spectrum., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)
- Published
- 2021
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44. Microchromosomes are building blocks of bird, reptile, and mammal chromosomes.
- Author
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Waters PD, Patel HR, Ruiz-Herrera A, Álvarez-González L, Lister NC, Simakov O, Ezaz T, Kaur P, Frere C, Grützner F, Georges A, and Graves JAM
- Subjects
- Animals, Base Sequence, Conserved Sequence, Biological Evolution, Chordata genetics, Chromosomes, Mammalian, Genome
- Abstract
Microchromosomes, once considered unimportant shreds of the chicken genome, are gene-rich elements with a high GC content and few transposable elements. Their origin has been debated for decades. We used cytological and whole-genome sequence comparisons, and chromosome conformation capture, to trace their origin and fate in genomes of reptiles, birds, and mammals. We find that microchromosomes as well as macrochromosomes are highly conserved across birds and share synteny with single small chromosomes of the chordate amphioxus, attesting to their origin as elements of an ancient animal genome. Turtles and squamates (snakes and lizards) share different subsets of ancestral microchromosomes, having independently lost microchromosomes by fusion with other microchromosomes or macrochromosomes. Patterns of fusions were quite different in different lineages. Cytological observations show that microchromosomes in all lineages are spatially separated into a central compartment at interphase and during mitosis and meiosis. This reflects higher interaction between microchromosomes than with macrochromosomes, as observed by chromosome conformation capture, and suggests some functional coherence. In highly rearranged genomes fused microchromosomes retain most ancestral characteristics, but these may erode over evolutionary time; surprisingly, de novo microchromosomes have rapidly adopted high interaction. Some chromosomes of early-branching monotreme mammals align to several bird microchromosomes, suggesting multiple microchromosome fusions in a mammalian ancestor. Subsequently, multiple rearrangements fueled the extraordinary karyotypic diversity of therian mammals. Thus, microchromosomes, far from being aberrant genetic elements, represent fundamental building blocks of amniote chromosomes, and it is mammals, rather than reptiles and birds, that are atypical., Competing Interests: Competing interest statement: Four authors (J.A.M.G., A.R.-H., T.E., and F.G.), as well as one of the referees, made contributions to a review of the continued usefulness of cytogenetics in the age of genomics, solicited and edited by Janine Deakin (https://www.mdpi.com/2073-4425/10/8/627)., (Copyright © 2021 the Author(s). Published by PNAS.)
- Published
- 2021
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45. Mechanistic insights into plant SUVH family H3K9 methyltransferases and their binding to context-biased non-CG DNA methylation
- Author
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Zhenhui Zhong, Jiamu Du, Rui Liu, C.J. Harris, Zonghua Wang, Baoqian Jia, Steven E. Jacobsen, Sisi Li, Xuezhen Li, and Wei Chen
- Subjects
0301 basic medicine ,Models, Molecular ,Methyltransferase ,Protein Conformation ,1.1 Normal biological development and functioning ,Arabidopsis ,Genetically Modified ,plant ,Crystallography, X-Ray ,Methylation ,H3K9me2 ,Histones ,03 medical and health sciences ,chemistry.chemical_compound ,Models ,Underpinning research ,Genetics ,SUVH6 ,Regulation of gene expression ,Cofactor binding ,Multidisciplinary ,Crystallography ,DNA methylation ,biology ,Base Sequence ,Chemistry ,Arabidopsis Proteins ,histone methyltransferase ,Lysine ,Human Genome ,Molecular ,DNA ,Histone-Lysine N-Methyltransferase ,Plants ,DNA Methylation ,Plants, Genetically Modified ,030104 developmental biology ,Histone ,PNAS Plus ,Histone methyltransferase ,biology.protein ,X-Ray ,Nucleic Acid Conformation ,Generic health relevance ,Protein Binding - Abstract
DNA methylation functions in gene silencing and the maintenance of genome integrity. In plants, non-CG DNA methylation is linked through a self-reinforcing loop with histone 3 lysine 9 dimethylation (H3K9me2). The plant-specific SUPPRESSOR OF VARIEGATION 3–9 HOMOLOG (SUVH) family H3K9 methyltransferases (MTases) bind to DNA methylation marks and catalyze H3K9 methylation. Here, we analyzed the structure and function of Arabidopsis thaliana SUVH6 to understand how this class of enzyme maintains methylation patterns in the genome. We reveal that SUVH6 has a distinct 5-methyl-dC (5mC) base-flipping mechanism involving a thumb loop element. Autoinhibition of H3 substrate entry is regulated by a SET domain loop, and a conformational transition in the post-SET domain upon cofactor binding may control catalysis. In vitro DNA binding and in vivo ChIP-seq data reveal that the different SUVH family H3K9 MTases have distinct DNA binding preferences, targeting H3K9 methylation to sites with different methylated DNA sequences, explaining the context biased non-CG DNA methylation in plants.
- Published
- 2018
46. Reduction in adaptor amounts establishes degradation hierarchy among protease substrates
- Author
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Xiaohui Gao, Eduardo A. Groisman, and Jinki Yeom
- Subjects
0301 basic medicine ,Proteases ,Protein subunit ,medicine.medical_treatment ,Proteolysis ,Sequence Homology ,medicine.disease_cause ,Substrate Specificity ,03 medical and health sciences ,Bacterial Proteins ,Transcription (biology) ,Salmonella ,medicine ,Escherichia coli ,Regulation of gene expression ,Multidisciplinary ,Protease ,medicine.diagnostic_test ,Base Sequence ,Chemistry ,Escherichia coli Proteins ,Cell biology ,030104 developmental biology ,PNAS Plus ,FtsA ,Carrier Proteins - Abstract
ATP-dependent proteases control critical cellular processes, including development, physiology, and virulence. A given protease may recognize a substrate directly via an unfoldase domain or subunit or indirectly via an adaptor that delivers the substrate to the unfoldase. We now report that cells achieve differential stability among substrates of a given protease by modulating adaptor amounts. We establish that the regulatory protein PhoP represses transcription of the gene specifying the ClpAP protease adaptor ClpS when the bacteria Salmonella enterica and Escherichia coli experience low cytoplasmic Mg2+. The resulting decrease in ClpS amounts diminishes proteolysis of several ClpSAP-dependent substrates, including the putrescine aminotransferase Oat, which heightens the formation of antibiotic persisters, and the transcriptional regulators UvrY and PhoP, which alter the expression of genes controlled by these proteins. By contrast, the decrease in ClpS amounts did not impact the abundance of the ClpSAP substrate FtsA, reflecting that FtsA binds to ClpS more tightly than to UvrY and PhoP. Our findings show how physiological conditions that reduce adaptor amounts modify the abundance of selected substrates of a given protease.
- Published
- 2018
47. Correction for Holmes et al., Altered metabotropic glutamate receptor 5 markers in PTSD: In vivo and postmortem evidence
- Subjects
Adult ,Male ,Base Sequence ,Sequence Analysis, RNA ,Receptor, Metabotropic Glutamate 5 ,Gene Expression ,Glutamic Acid ,Nerve Tissue Proteins ,Corrections ,Stress Disorders, Post-Traumatic ,Tacrolimus Binding Proteins ,Positron-Emission Tomography ,Humans ,Female ,Radiopharmaceuticals ,Glucocorticoids - Abstract
Posttraumatic stress disorder (PTSD) is a prevalent and highly disabling disorder, but there is currently no targeted pharmacological treatment for it. Dysfunction of the glutamate system has been implicated in trauma and stress psychopathology, resulting in a growing interest in modulation of the glutamate system for the treatment of PTSD. Specifically, the metabotropic glutamate receptor 5 (mGluR5) represents a promising treatment target. We used [
- Published
- 2017
48. Ciliopathy genes are required for apical secretion of Cochlin, an otolith crystallization factor.
- Author
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Leventea E, Zhu Z, Fang X, Nikolaeva Y, Markham E, Hirst RA, van Eeden FJM, and Malicki JJ
- Subjects
- Amino Acid Sequence, Animals, Bardet-Biedl Syndrome genetics, Base Sequence, Cilia metabolism, Crystallization, Epistasis, Genetic, Extracellular Matrix Proteins genetics, Gene Expression Regulation, Developmental, Homozygote, Mutation genetics, Phenotype, Zebrafish Proteins genetics, Ciliopathies genetics, Otolithic Membrane metabolism, Zebrafish genetics, Zebrafish Proteins metabolism
- Abstract
Here, we report that important regulators of cilia formation and ciliary compartment-directed protein transport function in secretion polarity. Mutations in cilia genes cep290 and bbs2 , involved in human ciliopathies, affect apical secretion of Cochlin, a major otolith component and a determinant of calcium carbonate crystallization form. We show that Cochlin, defective in human auditory and vestibular disorder, DFNA9, is secreted from small specialized regions of vestibular system epithelia. Cells of these regions secrete Cochlin both apically into the ear lumen and basally into the basal lamina. Basally secreted Cochlin diffuses along the basal surface of vestibular epithelia, while apically secreted Cochlin is incorporated into the otolith. Mutations in a subset of ciliopathy genes lead to defects in Cochlin apical secretion, causing abnormal otolith crystallization and behavioral defects. This study reveals a class of ciliary proteins that are important for the polarity of secretion and delineate a secretory pathway that regulates biomineralization., Competing Interests: The authors declare no competing interest.
- Published
- 2021
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49. Promoter-sequence determinants and structural basis of primer-dependent transcription initiation in Escherichia coli .
- Author
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Skalenko KS, Li L, Zhang Y, Vvedenskaya IO, Winkelman JT, Cope AL, Taylor DM, Shah P, Ebright RH, Kinney JB, Zhang Y, and Nickels BE
- Subjects
- Base Sequence, Binding Sites, Chromosomes, Bacterial genetics, Gene Expression Regulation, Bacterial, RNA, Messenger genetics, RNA, Messenger metabolism, Transcription Initiation Site, DNA Primers metabolism, Escherichia coli genetics, Promoter Regions, Genetic, Transcription Initiation, Genetic
- Abstract
Chemical modifications of RNA 5'-ends enable "epitranscriptomic" regulation, influencing multiple aspects of RNA fate. In transcription initiation, a large inventory of substrates compete with nucleoside triphosphates for use as initiating entities, providing an ab initio mechanism for altering the RNA 5'-end. In Escherichia coli cells, RNAs with a 5'-end hydroxyl are generated by use of dinucleotide RNAs as primers for transcription initiation, "primer-dependent initiation." Here, we use massively systematic transcript end readout (MASTER) to detect and quantify RNA 5'-ends generated by primer-dependent initiation for ∼4
10 (∼1,000,000) promoter sequences in E. coli The results show primer-dependent initiation in E. coli involves any of the 16 possible dinucleotide primers and depends on promoter sequences in, upstream, and downstream of the primer binding site. The results yield a consensus sequence for primer-dependent initiation, YTSS-2 NTSS-1 NTSS WTSS+1 , where TSS is the transcription start site, NTSS-1 NTSS is the primer binding site, Y is pyrimidine, and W is A or T. Biochemical and structure-determination studies show that the base pair (nontemplate-strand base:template-strand base) immediately upstream of the primer binding site (Y:RTSS-2 , where R is purine) exerts its effect through the base on the DNA template strand (RTSS-2 ) through interchain base stacking with the RNA primer. Results from analysis of a large set of natural, chromosomally encoded E coli promoters support the conclusions from MASTER. Our findings provide a mechanistic and structural description of how TSS-region sequence hard-codes not only the TSS position but also the potential for epitranscriptomic regulation through primer-dependent transcription initiation., Competing Interests: The authors declare no competing interest.- Published
- 2021
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50. Polymorphic tandem DNA repeats activate the human telomerase reverse transcriptase gene.
- Author
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Xu T, Cheng, Zhao Y, Zhang J, Zhu X, Zhang F, Chen G, Wang Y, Yan X, Robertson GP, Gaddameedhi S, Lazarus P, Wang S, and Zhu J
- Subjects
- Black or African American genetics, Aged, 80 and over, Animals, Base Sequence, Cell Differentiation genetics, Cell Line, Cell Proliferation genetics, Chromosomes, Artificial, Bacterial genetics, E-Box Elements genetics, Genome, Human, Human Embryonic Stem Cells metabolism, Humans, Mice, Nude, Neoplasms genetics, Neoplasms pathology, Promoter Regions, Genetic, Protein Binding genetics, Sequence Deletion genetics, Telomere Homeostasis genetics, Mice, Minisatellite Repeats genetics, Polymorphism, Genetic, Telomerase genetics, Transcriptional Activation
- Abstract
Multiple independent sequence variants of the hTERT locus have been associated with telomere length and cancer risks in genome-wide association studies. Here, we identified an intronic variable number tandem repeat, VNTR2-1, as an enhancer-like element, which activated hTERT transcription in a cell in a chromatin-dependent manner. VNTR2-1, consisting of 42-bp repeats with an array of enhancer boxes, cooperated with the proximal promoter in the regulation of hTERT transcription by basic helix-loop-helix transcription factors and maintained hTERT expression during embryonic stem-cell differentiation. Genomic deletion of VNTR2-1 in MelJuSo melanoma cells markedly reduced hTERT transcription, leading to telomere shortening, cellular senescence, and impairment of xenograft tumor growth. Interestingly, VNTR2-1 lengths varied widely in human populations; hTERT alleles with shorter VNTR2-1 were underrepresented in African American centenarians, indicating its role in human aging. Therefore, this polymorphic element is likely a missing link in the telomerase regulatory network and a molecular basis for genetic diversities of telomere homeostasis and age-related disease susceptibilities., Competing Interests: The authors declare no competing interest.
- Published
- 2021
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