International Journal of Molecular Sciences

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Many Faces of Next-Generation Sequencing in Gene Expression Studies

by Alexey A. Malygin

Int. J. Mol. Sci. 2023, 24(4), 4075; https://doi.org/10.3390/ijms24044075 - 17 Feb 2023

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Abstract

Gene expression is a fundamental cellular process that ensures the transfer of information encoded in a gene into the final functional product [...] Full article

(This article belongs to the Special Issue Regulation of Gene Expression in the NGS Era)

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17 pages, 3297 KiB

Open AccessArticle

Changes in the Transcriptome Caused by Mutations in the Ribosomal Protein uS10 Associated with a Predisposition to Colorectal Cancer

by Yueming Tian, Elena S. Babaylova, Alexander V. Gopanenko, Alexey E. Tupikin, Marsel R. Kabilov, Alexey A. Malygin and Galina G. Karpova

Int. J. Mol. Sci. 2022, 23(11), 6174; https://doi.org/10.3390/ijms23116174 - 31 May 2022

Cited by 3 | Viewed by 2211

Abstract

A number of mutations in the RPS20 gene encoding the ribosomal protein uS10 have been found to be associated with a predisposition to hereditary non-polyposis colorectal carcinoma (CRC). We transfected HEK293T cells with constructs carrying the uS10 minigene with mutations identical to those [...] Read more.

A number of mutations in the RPS20 gene encoding the ribosomal protein uS10 have been found to be associated with a predisposition to hereditary non-polyposis colorectal carcinoma (CRC). We transfected HEK293T cells with constructs carrying the uS10 minigene with mutations identical to those mentioned above and examined the effects of the produced proteins on the cellular transcriptome. We showed that uS10 with mutations p.V50SfsX23 or p.L61EfsX11 cannot be incorporated into 40S ribosomal subunits, while the protein with the missense mutation p.V54L functionally replaces the respective endogenous protein in the 40S subunit assembly and the translation process. The comparison of RNA-seq data obtained from cells producing aberrant forms of uS10 with data for those producing the wild-type protein revealed overlapping sets of upregulated and downregulated differently expressed genes (DEGs) related to several pathways. Among the limited number of upregulated DEGs, there were genes directly associated with the progression of CRC, e.g., PPM1D and PIGN. Our findings indicate that the accumulation of the mutant forms of uS10 triggers a cascade of cellular events, similar to that which is triggered when the cell responds to a large number of erroneous proteins, suggesting that this may increase the risk of cancer. Full article

(This article belongs to the Special Issue Regulation of Gene Expression in the NGS Era)

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12 pages, 1693 KiB

Open AccessArticle

Circulating miR-499a-5p Is a Potential Biomarker of MYH7—Associated Hypertrophic Cardiomyopathy

by Natalia Baulina, Maria Pisklova, Ivan Kiselev, Olga Chumakova, Dmitry Zateyshchikov and Olga Favorova

Int. J. Mol. Sci. 2022, 23(7), 3791; https://doi.org/10.3390/ijms23073791 - 30 Mar 2022

Cited by 10 | Viewed by 2458

Abstract

Hypertrophic cardiomyopathy (HCM) is the most common inherited myocardial disease with significant genetic and phenotypic heterogeneity. To search for novel biomarkers, which could increase the accuracy of HCM diagnosis and improve understanding of its phenotype formation, we analyzed the levels of circulating miRNAs—stable [...] Read more.

Hypertrophic cardiomyopathy (HCM) is the most common inherited myocardial disease with significant genetic and phenotypic heterogeneity. To search for novel biomarkers, which could increase the accuracy of HCM diagnosis and improve understanding of its phenotype formation, we analyzed the levels of circulating miRNAs—stable non-coding RNAs involved in post-transcriptional gene regulation. Performed high throughput sequencing of miRNAs in plasma of HCM patients and controls pinpointed miR-499a-5p as one of 35 miRNAs dysregulated in HCM. Further investigation on enlarged groups of individuals showed that its level was higher in carriers of pathogenic/likely pathogenic (P/LP) variants in MYH7 gene compared to controls (fold change, FC = 8.9; p < 0.0001). Just as important, carriers of variants in MYH7 gene were defined with higher miRNA levels than carriers of variants in the MYBPC3 gene (FC = 14.1; p = 0.0003) and other patients (FC = 4.1; p = 0.0008). The receiver operating characteristic analysis analysis showed the ability of miR-499a-5p to identify MYH7 variant carriers with the HCM phenotype with area under the curve value of 0.95 (95% confidence interval: 0.88–1.03, p = 0.0004); sensitivity and specificity were 0.86 and 0.91 (cut-off = 0.0014). Therefore, miR-499a-5p could serve as a circulating biomarker of HCM, caused by P/LP variants in MYH7 gene. Full article

(This article belongs to the Special Issue Regulation of Gene Expression in the NGS Era)

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41 pages, 7384 KiB

Open AccessArticle

Combined Transcriptomic and Proteomic Profiling of E. coli under Microaerobic versus Aerobic Conditions: The Multifaceted Roles of Noncoding Small RNAs and Oxygen-Dependent Sensing in Global Gene Expression Control

by Gunn-Guang Liou, Anna Chao Kaberdina, Wei-Syuan Wang, Vladimir R. Kaberdin and Sue Lin-Chao

Int. J. Mol. Sci. 2022, 23(5), 2570; https://doi.org/10.3390/ijms23052570 - 25 Feb 2022

Cited by 6 | Viewed by 3896

Abstract

Adaptive mechanisms that facilitate intestinal colonization by the human microbiota, including Escherichia coli, may be better understood by analyzing the physiology and gene expression of bacteria in low-oxygen environments. We used high-throughput transcriptomics and proteomics to compare the expression profiles of E. [...] Read more.

Adaptive mechanisms that facilitate intestinal colonization by the human microbiota, including Escherichia coli, may be better understood by analyzing the physiology and gene expression of bacteria in low-oxygen environments. We used high-throughput transcriptomics and proteomics to compare the expression profiles of E. coli grown under aerobic versus microaerobic conditions. Clustering of high-abundance transcripts under microaerobiosis highlighted genes controlling acid-stress adaptation (gadAXW, gadAB, hdeAB-yhiD and hdeD operons), cell adhesion/biofilm formation (pgaABCD and csgDEFG operons), electron transport (cydAB), oligopeptide transport (oppABCDF), and anaerobic respiration/fermentation (hyaABCDEF and hycABCDEFGHI operons). In contrast, downregulated genes were involved in iron transport (fhuABCD, feoABC and fepA-entD operons), iron-sulfur cluster assembly (iscRSUA and sufABCDSE operons), aerobic respiration (sdhDAB and sucABCDSE operons), and de novo nucleotide synthesis (nrdHIEF). Additionally, quantitative proteomics showed that the products (proteins) of these high- or low-abundance transcripts were expressed consistently. Our findings highlight interrelationships among energy production, carbon metabolism, and iron homeostasis. Moreover, we have identified and validated a subset of differentially expressed noncoding small RNAs (i.e., CsrC, RyhB, RprA and GcvB), and we discuss their regulatory functions during microaerobic growth. Collectively, we reveal key changes in gene expression at the transcriptional and post-transcriptional levels that sustain E. coli growth when oxygen levels are low. Full article

(This article belongs to the Special Issue Regulation of Gene Expression in the NGS Era)

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19 pages, 14498 KiB

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The Functional Consequences of the Novel Ribosomal Pausing Site in SARS-CoV-2 Spike Glycoprotein RNA

by Olga A. Postnikova, Sheetal Uppal, Weiliang Huang, Maureen A. Kane, Rafael Villasmil, Igor B. Rogozin, Eugenia Poliakov and T. Michael Redmond

Int. J. Mol. Sci. 2021, 22(12), 6490; https://doi.org/10.3390/ijms22126490 - 17 Jun 2021

Cited by 15 | Viewed by 7521

Abstract

The SARS-CoV-2 Spike glycoprotein (S protein) acquired a unique new 4 amino acid -PRRA- insertion sequence at amino acid residues (aa) 681–684 that forms a new furin cleavage site in S protein as well as several new glycosylation sites. We studied various statistical [...] Read more.

The SARS-CoV-2 Spike glycoprotein (S protein) acquired a unique new 4 amino acid -PRRA- insertion sequence at amino acid residues (aa) 681–684 that forms a new furin cleavage site in S protein as well as several new glycosylation sites. We studied various statistical properties of the -PRRA- insertion at the RNA level (CCUCGGCGGGCA). The nucleotide composition and codon usage of this sequence are different from the rest of the SARS-CoV-2 genome. One of such features is two tandem CGG codons, although the CGG codon is the rarest codon in the SARS-CoV-2 genome. This suggests that the insertion sequence could cause ribosome pausing as the result of these rare codons. Due to population variants, the Nextstrain divergence measure of the CCU codon is extremely large. We cannot exclude that this divergence might affect host immune responses/effectiveness of SARS-CoV-2 vaccines, possibilities awaiting further investigation. Our experimental studies show that the expression level of original RNA sequence “wildtype” spike protein is much lower than for codon-optimized spike protein in all studied cell lines. Interestingly, the original spike sequence produces a higher titer of pseudoviral particles and a higher level of infection. Further mutagenesis experiments suggest that this dual-effect insert, comprised of a combination of overlapping translation pausing and furin sites, has allowed SARS-CoV-2 to infect its new host (human) more readily. This underlines the importance of ribosome pausing to allow efficient regulation of protein expression and also of cotranslational subdomain folding. Full article

(This article belongs to the Special Issue Regulation of Gene Expression in the NGS Era)

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16 pages, 2488 KiB

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Knockdown of the Ribosomal Protein eL38 in HEK293 Cells Changes the Translational Efficiency of Specific Genes

by Alexander V. Gopanenko, Alena V. Kolobova, Alexey E. Tupikin, Marsel R. Kabilov, Alexey A. Malygin and Galina G. Karpova

Int. J. Mol. Sci. 2021, 22(9), 4531; https://doi.org/10.3390/ijms22094531 - 26 Apr 2021

Cited by 4 | Viewed by 3066

Abstract

The protein eL38 is one of the smallest proteins of the mammalian ribosome, which is a component of its large (60S) subunit. The haploinsufficiency of eL38 in mice leads to the Tail-short mutant phenotype characterized by defects in the development of the axial [...] Read more.

The protein eL38 is one of the smallest proteins of the mammalian ribosome, which is a component of its large (60S) subunit. The haploinsufficiency of eL38 in mice leads to the Tail-short mutant phenotype characterized by defects in the development of the axial skeleton caused by the poor translation of mRNA subsets of Hox genes. Using the ribosome profiling assay applied to HEK293 cells knocked down of eL38, we examined the effects of the lack of eL38 in 60S subunits on gene expression at the level of translation. A four-fold decrease in the cell content of eL38 was shown to result in significant changes in the translational efficiencies of 150 genes. Among the genes, whose expression at the level of translation was enhanced, there were mainly those associated with basic metabolic processes; namely, translation, protein folding, chromosome organization, splicing, and others. The set of genes with reduced translation efficiencies contained those that are mostly involved in the processes related to the regulation of transcription, including the activation of Hox genes. Thus, we demonstrated that eL38 insufficiency significantly affects the expression of certain genes at the translational level. Our findings facilitate understanding the possible causes of some anomalies in eL38-deficient animals. Full article

(This article belongs to the Special Issue Regulation of Gene Expression in the NGS Era)

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27 pages, 5445 KiB

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The MarR-Type Regulator PA3458 Is Involved in Osmoadaptation Control in Pseudomonas aeruginosa

by Karolina Kotecka, Adam Kawalek, Kamil Kobylecki and Aneta Agnieszka Bartosik

Int. J. Mol. Sci. 2021, 22(8), 3982; https://doi.org/10.3390/ijms22083982 - 12 Apr 2021

Cited by 11 | Viewed by 3206

Abstract

Pseudomonas aeruginosa is a facultative human pathogen, causing acute and chronic infections that are especially dangerous for immunocompromised patients. The eradication of P. aeruginosa is difficult due to its intrinsic antibiotic resistance mechanisms, high adaptability, and genetic plasticity. The bacterium possesses multilevel regulatory [...] Read more.

Pseudomonas aeruginosa is a facultative human pathogen, causing acute and chronic infections that are especially dangerous for immunocompromised patients. The eradication of P. aeruginosa is difficult due to its intrinsic antibiotic resistance mechanisms, high adaptability, and genetic plasticity. The bacterium possesses multilevel regulatory systems engaging a huge repertoire of transcriptional regulators (TRs). Among these, the MarR family encompasses a number of proteins, mainly acting as repressors, which are involved in response to various environmental signals. In this work, we aimed to decipher the role of PA3458, a putative MarR-type TR from P. aeruginosa. Transcriptional profiling of P. aeruginosa PAO1161 overexpressing PA3458 showed changes in the mRNA level of 133 genes; among them, 100 were down-regulated, suggesting the repressor function of PA3458. Concomitantly, ChIP-seq analysis identified more than 300 PA3458 binding sites in P. aeruginosa. The PA3458 regulon encompasses genes involved in stress response, including the PA3459–PA3461 operon, which is divergent to PA3458. This operon encodes an asparagine synthase, a GNAT-family acetyltransferase, and a glutamyl aminopeptidase engaged in the production of N-acetylglutaminylglutamine amide (NAGGN), which is a potent bacterial osmoprotectant. We showed that PA3458-mediated control of PA3459–PA3461 expression is required for the adaptation of P. aeruginosa growth in high osmolarity. Overall, our data indicate that PA3458 plays a role in osmoadaptation control in P. aeruginosa. Full article

(This article belongs to the Special Issue Regulation of Gene Expression in the NGS Era)

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20 pages, 3875 KiB

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ceRNA Network Regulation of TGF-β, WNT, FOXO, Hedgehog Pathways in the Pharynx of Ciona robusta

by Aiti Vizzini, Angela Bonura, Laura La Paglia, Antonino Fiannaca, Massimo La Rosa, Alfonso Urso and Vincenzo Arizza

Int. J. Mol. Sci. 2021, 22(7), 3497; https://doi.org/10.3390/ijms22073497 - 28 Mar 2021

Cited by 6 | Viewed by 2521

Abstract

The transforming growth factor-β (TGF-β) family of cytokines performs a multifunctional signaling, which is integrated and coordinated in a signaling network that involves other pathways, such as Wintless, Forkhead box-O (FOXO) and Hedgehog and regulates pivotal functions related to cell fate in all [...] Read more.

The transforming growth factor-β (TGF-β) family of cytokines performs a multifunctional signaling, which is integrated and coordinated in a signaling network that involves other pathways, such as Wintless, Forkhead box-O (FOXO) and Hedgehog and regulates pivotal functions related to cell fate in all tissues. In the hematopoietic system, TGF-β signaling controls a wide spectrum of biological processes, from immune system homeostasis to the quiescence and self-renewal of hematopoietic stem cells (HSCs). Recently an important role in post-transcription regulation has been attributed to two type of ncRNAs: microRNAs and pseudogenes. Ciona robusta, due to its philogenetic position close to vertebrates, is an excellent model to investigate mechanisms of post-transcriptional regulation evolutionarily highly conserved in immune homeostasis. The combined use of NGS and bioinformatic analyses suggests that in the pharynx, the hematopoietic organ of Ciona robusta, the Tgf-β, Wnt, Hedgehog and FoxO pathways are involved in tissue homeostasis, as they are in human. Furthermore, ceRNA network interactions and 3′UTR elements analyses of Tgf-β, Wnt, Hedgehog and FoxO pathways genes suggest that different miRNAs conserved (cin-let-7d, cin-mir-92c, cin-mir-153), species-specific (cin-mir-4187, cin-mir-4011a, cin-mir-4056, cin-mir-4150, cin-mir-4189, cin-mir-4053, cin-mir-4016, cin-mir-4075), pseudogenes (ENSCING00000011392, ENSCING00000018651, ENSCING00000007698) and mRNA 3′UTR elements are involved in post-transcriptional regulation in an integrated way in C. robusta. Full article

(This article belongs to the Special Issue Regulation of Gene Expression in the NGS Era)

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Review

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14 pages, 757 KiB

Open AccessReview

Aging, Bone Marrow and Next-Generation Sequencing (NGS): Recent Advances and Future Perspectives

by Payal Ganguly, Bradley Toghill and Shelly Pathak

Int. J. Mol. Sci. 2021, 22(22), 12225; https://doi.org/10.3390/ijms222212225 - 12 Nov 2021

Cited by 11 | Viewed by 3920

Abstract

The aging of bone marrow (BM) remains a very imperative and alluring subject, with an ever-increasing interest among fellow scientists. A considerable amount of progress has been made in this field with the established ‘hallmarks of aging’ and continued efforts to investigate the [...] Read more.

The aging of bone marrow (BM) remains a very imperative and alluring subject, with an ever-increasing interest among fellow scientists. A considerable amount of progress has been made in this field with the established ‘hallmarks of aging’ and continued efforts to investigate the age-related changes observed within the BM. Inflammaging is considered as a low-grade state of inflammation associated with aging, and whilst the possible mechanisms by which aging occurs are now largely understood, the processes leading to the underlying changes within aged BM remain elusive. The ability to identify these changes and detect such alterations at the genetic level are key to broadening the knowledgebase of aging BM. Next-generation sequencing (NGS) is an important molecular-level application presenting the ability to not only determine genomic base changes but provide transcriptional profiling (RNA-seq), as well as a high-throughput analysis of DNA–protein interactions (ChIP-seq). Utilising NGS to explore the genetic alterations occurring over the aging process within alterative cell types facilitates the comprehension of the molecular and cellular changes influencing the dynamics of aging BM. Thus, this review prospects the current landscape of BM aging and explores how NGS technology is currently being applied within this ever-expanding field of research. Full article

(This article belongs to the Special Issue Regulation of Gene Expression in the NGS Era)

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