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13 pages, 3081 KiB

Open AccessArticle

Differential Gene Expression following DHX36/G4R1 Knockout Is Associated with G-Quadruplex Content and Cancer

by Joseph M. Gumina, Adam E. Richardson, Mahmudul Hasan Shojiv, Antonio E. Chambers, Siara N. Sandwith, Michael A. Reisinger, Taylor J. Karns, Tyler L. Osborne, Hasna N. Alashi, Quinn T. Anderson, Meredith E. Sharlow, Dylan C. Seiler, Evan M. Rogers, Anna R. Bartosik, Melissa A. Smaldino, James P. Vaughn, Yuh-Hwa Wang, Philip J. Smaldino and Robert A. Haney

Int. J. Mol. Sci. 2024, 25(3), 1753; https://doi.org/10.3390/ijms25031753 - 01 Feb 2024

Viewed by 784

Abstract

G-quadruplexes (G4s) are secondary DNA and RNA structures stabilized by positive cations in a central channel formed by stacked tetrads of Hoogsteen base-paired guanines. G4s form from G-rich sequences across the genome, whose biased distribution in regulatory regions points towards a gene-regulatory role. [...] Read more.

G-quadruplexes (G4s) are secondary DNA and RNA structures stabilized by positive cations in a central channel formed by stacked tetrads of Hoogsteen base-paired guanines. G4s form from G-rich sequences across the genome, whose biased distribution in regulatory regions points towards a gene-regulatory role. G4s can themselves be regulated by helicases, such as DHX36 (aliases: G4R1 and RHAU), which possess the necessary activity to resolve these stable structures. G4s have been shown to both positively and negatively regulate gene expression when stabilized by ligands, or through the loss of helicase activity. Using DHX36 knockout Jurkat cell lines, we identified widespread, although often subtle, effects on gene expression that are associated with the presence or number of observed G-quadruplexes in promoters or gene regions. Genes that significantly change their expression, particularly those that show a significant increase in RNA abundance under DHX36 knockout, are associated with a range of cellular functions and processes, including numerous transcription factors and oncogenes, and are linked to several cancers. Our work highlights the direct and indirect role of DHX36 in the transcriptome of T-lymphocyte leukemia cells and the potential for DHX36 dysregulation in cancer. Full article

(This article belongs to the Special Issue Bioinformatics of Unusual DNA and RNA Structures)

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24 pages, 7409 KiB

Open AccessArticle

The Fate and Functionality of Alien tRNA Fragments in Culturing Medium and Cells of Escherichia coli

by Konstantin S. Shavkunov, Natalia Yu. Markelova, Olga A. Glazunova, Nikolay P. Kolzhetsov, Valery V. Panyukov and Olga N. Ozoline

Int. J. Mol. Sci. 2023, 24(16), 12960; https://doi.org/10.3390/ijms241612960 - 19 Aug 2023

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Abstract

Numerous observations have supported the idea that various types of noncoding RNAs, including tRNA fragments (tRFs), are involved in communications between the host and its microbial community. The possibility of using their signaling function has stimulated the study of secreted RNAs, potentially involved [...] Read more.

Numerous observations have supported the idea that various types of noncoding RNAs, including tRNA fragments (tRFs), are involved in communications between the host and its microbial community. The possibility of using their signaling function has stimulated the study of secreted RNAs, potentially involved in the interspecies interaction of bacteria. This work aimed at identifying such RNAs and characterizing their maturation during transport. We applied an approach that allowed us to detect oligoribonucleotides secreted by Prevotella copri (Segatella copri) or Rhodospirillum rubrum inside Escherichia coli cells. Four tRFs imported by E. coli cells co-cultured with these bacteria were obtained via chemical synthesis, and all of them affected the growth of E. coli. Their successive modifications in the culture medium and recipient cells were studied by high-throughput cDNA sequencing. Instead of the expected accidental exonucleolysis, in the milieu, we observed nonrandom cleavage by endonucleases continued in recipient cells. We also found intramolecular rearrangements of synthetic oligonucleotides, which may be considered traces of intermediate RNA circular isomerization. Using custom software, we estimated the frequency of such events in transcriptomes and secretomes of E. coli and observed surprising reproducibility in positions of such rare events, assuming the functionality of ring isoforms or their permuted derivatives in bacteria. Full article

(This article belongs to the Special Issue Bioinformatics of Unusual DNA and RNA Structures)

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19 pages, 2184 KiB

Open AccessArticle

In Silico Identification of Potential Quadruplex Forming Sequences in LncRNAs of Cervical Cancer

by Deepshikha Singh, Nakshi Desai, Viraj Shah and Bhaskar Datta

Int. J. Mol. Sci. 2023, 24(16), 12658; https://doi.org/10.3390/ijms241612658 - 10 Aug 2023

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Abstract

Long non-coding RNAs (lncRNAs) have emerged as auxiliary regulators of gene expression influencing tumor microenvironment, metastasis and radio-resistance in cancer. The presence of lncRNA in extracellular fluids makes them promising diagnostic markers. LncRNAs deploy higher-order structures to facilitate a complex range of functions. [...] Read more.

Long non-coding RNAs (lncRNAs) have emerged as auxiliary regulators of gene expression influencing tumor microenvironment, metastasis and radio-resistance in cancer. The presence of lncRNA in extracellular fluids makes them promising diagnostic markers. LncRNAs deploy higher-order structures to facilitate a complex range of functions. Among such structures, G-quadruplexes (G4s) can be detected or targeted by small molecular probes to drive theranostic applications. The in vitro identification of G4 formation in lncRNAs can be a tedious and expensive proposition. Bioinformatics-driven strategies can provide comprehensive and economic alternatives in conjunction with suitable experimental validation. We propose a pipeline to identify G4-forming sequences, protein partners and biological functions associated with dysregulated lncRNAs in cervical cancer. We identified 17 lncRNA clusters which possess transcripts that can fold into a G4 structure. We confirmed in vitro G4 formation in the four biologically active isoforms of SNHG20, MEG3, CRNDE and LINP1 by Circular Dichroism spectroscopy and Thioflavin-T-assisted fluorescence spectroscopy and reverse-transcriptase stop assay. Gene expression data demonstrated that these four lncRNAs can be potential prognostic biomarkers of cervical cancer. Two approaches were employed for identifying G4 specific protein partners for these lncRNAs and FMR2 was a potential interacting partner for all four clusters. We report a detailed investigation of G4 formation in lncRNAs that are dysregulated in cervical cancer. LncRNAs MEG3, CRNDE, LINP1 and SNHG20 are shown to influence cervical cancer progression and we report G4 specific protein partners for these lncRNAs. The protein partners and G4s predicted in lncRNAs can be exploited for theranostic objectives. Full article

(This article belongs to the Special Issue Bioinformatics of Unusual DNA and RNA Structures)

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14 pages, 1450 KiB

Open AccessArticle

Presence, Location and Conservation of Putative G-Quadruplex Forming Sequences in Arboviruses Infecting Humans

by Giulia Nicoletto, Sara N. Richter and Ilaria Frasson

Int. J. Mol. Sci. 2023, 24(11), 9523; https://doi.org/10.3390/ijms24119523 - 30 May 2023

Cited by 1 | Viewed by 1187

Abstract

Guanine quadruplexes (G4s) are non-canonical nucleic acid structures formed by guanine (G)-rich tracts that assemble into a core of stacked planar tetrads. G4s are found in the human genome and in the genomes of human pathogens, where they are involved in the regulation [...] Read more.

Guanine quadruplexes (G4s) are non-canonical nucleic acid structures formed by guanine (G)-rich tracts that assemble into a core of stacked planar tetrads. G4s are found in the human genome and in the genomes of human pathogens, where they are involved in the regulation of gene expression and genome replication. G4s have been proposed as novel pharmacological targets in humans and their exploitation for antiviral therapy is an emerging research topic. Here, we report on the presence, conservation and localization of putative G4-forming sequences (PQSs) in human arboviruses. The prediction of PQSs was performed on more than twelve thousand viral genomes, belonging to forty different arboviruses that infect humans, and revealed that the abundance of PQSs in arboviruses is not related to the genomic GC content, but depends on the type of nucleic acid that constitutes the viral genome. Positive-strand ssRNA arboviruses, especially Flaviviruses, are significantly enriched in highly conserved PQSs, located in coding sequences (CDSs) or untranslated regions (UTRs). In contrast, negative-strand ssRNA and dsRNA arboviruses contain few conserved PQSs. Our analyses also revealed the presence of bulged PQSs, accounting for 17–26% of the total predicted PQSs. The data presented highlight the presence of highly conserved PQS in human arboviruses and present non-canonical nucleic acid-structures as promising therapeutic targets in arbovirus infections. Full article

(This article belongs to the Special Issue Bioinformatics of Unusual DNA and RNA Structures)

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22 pages, 3650 KiB

Open AccessArticle

Structural Polymorphism of Guanine Quadruplex-Containing Regions in Human Promoters

by Christopher Hennecker, Lynn Yamout, Chuyang Zhang, Chenzhi Zhao, David Hiraki, Nicolas Moitessier and Anthony Mittermaier

Int. J. Mol. Sci. 2022, 23(24), 16020; https://doi.org/10.3390/ijms232416020 - 16 Dec 2022

Cited by 4 | Viewed by 1671

Abstract

Intramolecular guanine quadruplexes (G4s) are non-canonical nucleic acid structures formed by four guanine (G)-rich tracts that assemble into a core of stacked planar tetrads. G4-forming DNA sequences are enriched in gene promoters and are implicated in the control of gene expression. Most G4-forming [...] Read more.

Intramolecular guanine quadruplexes (G4s) are non-canonical nucleic acid structures formed by four guanine (G)-rich tracts that assemble into a core of stacked planar tetrads. G4-forming DNA sequences are enriched in gene promoters and are implicated in the control of gene expression. Most G4-forming DNA contains more G residues than can simultaneously be incorporated into the core resulting in a variety of different possible G4 structures. Although this kind of structural polymorphism is well recognized in the literature, there remain unanswered questions regarding possible connections between G4 polymorphism and biological function. Here we report a detailed bioinformatic survey of G4 polymorphism in human gene promoter regions. Our analysis is based on identifying G4-containing regions (G4CRs), which we define as stretches of DNA in which every residue can form part of a G4. We found that G4CRs with higher degrees of polymorphism are more tightly clustered near transcription sites and tend to contain G4s with shorter loops and bulges. Furthermore, we found that G4CRs with well-characterized biological functions tended to be longer and more polymorphic than genome-wide averages. These results represent new evidence linking G4 polymorphism to biological function and provide new criteria for identifying biologically relevant G4-forming regions from genomic data. Full article

(This article belongs to the Special Issue Bioinformatics of Unusual DNA and RNA Structures)

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15 pages, 4169 KiB

Open AccessArticle

Epigenomic Features and Potential Functions of K⁺ and Na⁺ Favorable DNA G-Quadruplexes in Rice

by Yilong Feng, Zhenyu Luo, Ranran Huang, Xueming Yang, Xuejiao Cheng and Wenli Zhang

Int. J. Mol. Sci. 2022, 23(15), 8404; https://doi.org/10.3390/ijms23158404 - 29 Jul 2022

Cited by 2 | Viewed by 1859

Abstract

DNA G-quadruplexes (G4s) are non-canonical four-stranded DNA structures involved in various biological processes in eukaryotes. Molecularly crowded solutions and monovalent cations have been reported to stabilize in vitro and in vivo G4 formation. However, how K⁺ and Na⁺ affect G4 formation [...] Read more.

DNA G-quadruplexes (G4s) are non-canonical four-stranded DNA structures involved in various biological processes in eukaryotes. Molecularly crowded solutions and monovalent cations have been reported to stabilize in vitro and in vivo G4 formation. However, how K⁺ and Na⁺ affect G4 formation genome-wide is still unclear in plants. Here, we conducted BG4-DNA-IP-seq, DNA immunoprecipitation with anti-BG4 antibody coupled with sequencing, under K⁺ and Na⁺ + PEG conditions in vitro. We found that K⁺-specific IP-G4s had a longer peak size, more GC and PQS content, and distinct AT and GC skews compared to Na⁺-specific IP-G4s. Moreover, K⁺- and Na⁺-specific IP-G4s exhibited differential subgenomic enrichment and distinct putative functional motifs for the binding of certain trans-factors. More importantly, we found that K⁺-specific IP-G4s were more associated with active marks, such as active histone marks, and low DNA methylation levels, as compared to Na⁺-specific IP-G4s; thus, K⁺-specific IP-G4s in combination with active chromatin features facilitate the expression of overlapping genes. In addition, K⁺- and Na⁺-specific IP-G4 overlapping genes exhibited differential GO (gene ontology) terms, suggesting they may have distinct biological relevance in rice. Thus, our study, for the first time, explores the effects of K⁺ and Na⁺ on global G4 formation in vitro, thereby providing valuable resources for functional G4 studies in rice. It will provide certain G4 loci for the biotechnological engineering of rice in the future. Full article

(This article belongs to the Special Issue Bioinformatics of Unusual DNA and RNA Structures)

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8 pages, 1185 KiB

Open AccessArticle

R-Loop Tracker: Web Access-Based Tool for R-Loop Detection and Analysis in Genomic DNA Sequences

by Václav Brázda, Jan Havlík, Jan Kolomazník, Oldřich Trenz and Jiří Šťastný

Int. J. Mol. Sci. 2021, 22(23), 12857; https://doi.org/10.3390/ijms222312857 - 27 Nov 2021

Cited by 4 | Viewed by 2491

Abstract

R-loops are common non-B nucleic acid structures formed by a three-stranded nucleic acid composed of an RNA–DNA hybrid and a displaced single-stranded DNA (ssDNA) loop. Because the aberrant R-loop formation leads to increased mutagenesis, hyper-recombination, rearrangements, and transcription-replication collisions, it is regarded as [...] Read more.

R-loops are common non-B nucleic acid structures formed by a three-stranded nucleic acid composed of an RNA–DNA hybrid and a displaced single-stranded DNA (ssDNA) loop. Because the aberrant R-loop formation leads to increased mutagenesis, hyper-recombination, rearrangements, and transcription-replication collisions, it is regarded as important in human diseases. Therefore, its prevalence and distribution in genomes are studied intensively. However, in silico tools for R-loop prediction are limited, and therefore, we have developed the R-loop tracker tool, which was implemented as a part of the DNA Analyser web server. This new tool is focused upon (1) prediction of R-loops in genomic DNA without length and sequence limitations; (2) integration of R-loop tracker results with other tools for nucleic acids analyses, including Genome Browser; (3) internal cross-evaluation of in silico results with experimental data, where available; (4) easy export and correlation analyses with other genome features and markers; and (5) enhanced visualization outputs. Our new R-loop tracker tool is freely accessible on the web pages of DNA Analyser tools, and its implementation on the web-based server allows effective analyses not only for DNA segments but also for full chromosomes and genomes. Full article

(This article belongs to the Special Issue Bioinformatics of Unusual DNA and RNA Structures)

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Review

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17 pages, 2071 KiB

Open AccessReview

Virus-Induced Gene Silencing (VIGS): A Powerful Tool for Crop Improvement and Its Advancement towards Epigenetics

by Sumer Zulfiqar, Muhammad Awais Farooq, Tiantian Zhao, PeiPei Wang, Javaria Tabusam, Yanhua Wang, Shuxin Xuan, Jianjun Zhao, Xueping Chen, Shuxing Shen and Aixia Gu

Int. J. Mol. Sci. 2023, 24(6), 5608; https://doi.org/10.3390/ijms24065608 - 15 Mar 2023

Cited by 9 | Viewed by 6389

Abstract

Virus-induced gene silencing (VIGS) is an RNA-mediated reverse genetics technology that has evolved into an indispensable approach for analyzing the function of genes. It downregulates endogenous genes by utilizing the posttranscriptional gene silencing (PTGS) machinery of plants to prevent systemic viral infections. Based [...] Read more.

Virus-induced gene silencing (VIGS) is an RNA-mediated reverse genetics technology that has evolved into an indispensable approach for analyzing the function of genes. It downregulates endogenous genes by utilizing the posttranscriptional gene silencing (PTGS) machinery of plants to prevent systemic viral infections. Based on recent advances, VIGS can now be used as a high-throughput tool that induces heritable epigenetic modifications in plants through the viral genome by transiently knocking down targeted gene expression. As a result of the progression of DNA methylation induced by VIGS, new stable genotypes with desired traits are being developed in plants. In plants, RNA-directed DNA methylation (RdDM) is a mechanism where epigenetic modifiers are guided to target loci by small RNAs, which play a major role in the silencing of the target gene. In this review, we described the molecular mechanisms of DNA and RNA-based viral vectors and the knowledge obtained through altering the genes in the studied plants that are not usually accessible to transgenic techniques. We showed how VIGS-induced gene silencing can be used to characterize transgenerational gene function(s) and altered epigenetic marks, which can improve future plant breeding programs. Full article

(This article belongs to the Special Issue Bioinformatics of Unusual DNA and RNA Structures)

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