Tenascin C as a novel zinc finger protein 750 target regulating the immunogenicity via DNA damage in lung squamous cell carcinoma.

Modulation of DNA damage repair in lung squamous cell carcinoma (LUSC) can result in the generation of neoantigens and heightened immunogenicity. Therefore, understanding DNA damage repair mechanisms holds significant clinical relevance for identifying targets for immunotherapy and devising therapeutic strategies. Our research has unveiled that the tumor suppressor zinc finger protein 750 (ZNF750) in LUSC binds to the promoter region of tenascin C (TNC), leading to reduced TNC expression. This modulation may impact the malignant behavior of tumor cells and is associated with patient prognosis. Additionally, single-cell RNA sequencing (scRNA-seq) of LUSC tissues has demonstrated an inverse correlation between ZNF750/TNC expression levels and immunogenicity. Manipulation of the ZNF750-TNC axis in vitro within LUSC cells has shown differential sensitivity to CD8+ cells, underscoring its pivotal role in regulating cellular immunogenicity. Further transcriptome sequencing analysis, DNA damage repair assay, and single-strand break analyses have revealed the involvement of the ZNF750-TNC axis in determining the preference for homologous recombination (HR) repair or non-homologous end joining (NHEJ) repair of DNA damage. with involvement of the Hippo/ERK signaling pathway. In summary, this study sheds light on the ZNF750-TNC axis's role in DNA damage repair regulation in LUSC, laying a groundwork for future translational research in immune cell therapy for LUSC.

Common occurrence of hotspots of single strand DNA breaks at transcriptional start sites.

BACKGROUND: We recently developed two high-resolution methods for genome-wide mapping of two prominent types of DNA damage, single-strand DNA breaks (SSBs) and abasic (AP) sites and found highly complex and non-random patterns of these lesions in mammalian genomes. One salient feature of SSB and AP sites was the existence of single-nucleotide hotspots for both lesions. RESULTS: In this work, we show that SSB hotspots are enriched in the immediate vicinity of transcriptional start sites (TSSs) in multiple normal mammalian tissues, however the magnitude of enrichment varies significantly with tissue type and appears to be limited to a subset of genes. SSB hotspots around TSSs are enriched on the template strand and associate with higher expression of the corresponding genes. Interestingly, SSB hotspots appear to be at least in part generated by the base-excision repair (BER) pathway from the AP sites. CONCLUSIONS: Our results highlight complex relationship between DNA damage and regulation of gene expression and suggest an exciting possibility that SSBs at TSSs might function as sensors of DNA damage to activate genes important for DNA damage response.

Effects of Graphene-Based Nanomaterials on Microorganisms and Soil Microbial Communities.

The past decades have witnessed intensive research on the biological effects of graphene-based nanomaterials (GBNs) and the application of GBNs in different fields. The published literature shows that GBNs exhibit inhibitory effects on almost all microorganisms under pure culture conditions, and that this inhibitory effect is influenced by the microbial species, the GBN's physicochemical properties, the GBN's concentration, treatment time, and experimental surroundings. In addition, microorganisms exist in the soil in the form of microbial communities. Considering the complex interactions between different soil components, different microbial communities, and GBNs in the soil environment, the effects of GBNs on soil microbial communities are undoubtedly intertwined. Since bacteria and fungi are major players in terrestrial biogeochemistry, this review focuses on the antibacterial and antifungal performance of GBNs, their antimicrobial mechanisms and influencing factors, as well as the impact of this effect on soil microbial communities. This review will provide a better understanding of the effects of GBNs on microorganisms at both the individual and population scales, thus providing an ecologically safe reference for the release of GBNs to different soil environments.

Glucosinolate O-methyltransferase mediated callus formation and affected ROS homeostasis in Arabidopsis thaliana.

Auxin-induced callus formation was largely dependent on the function of Lateral Organ Boundaries Domain (LBD) family transcription factors. We previously revealed that two IGMT (Indole glucosinolate oxy-methyl transferase) genes, IGMT2 and IGMT3, may be involved in the callus formation process as potential target genes of LBD29. Overexpression of the IGMT genes induces spontaneous callus formation. However, the details of the IGMT involvement in callus formation process were not well studied. IGMT1-4, but not IGMT5, are targeted and induced by LBD29 during the early stage of callus formation. Cell membrane and nucleus localized IGMT3 was mainly expressed in the elongation and maturation zones tissues of the primary root and lateral root, which could be further accumulated after CIM treatment. The igmts quadruple mutant, which obtained by CRISPR/Cas9 technology, exhibits a phenotype of attenuated callus formation. Enhanced indole glucosinolate anabolic pathway caused by IGMT1-4 overexpression promotes callus formation. In addition, the IGMT genes were involved in the reactive oxygen species homeostasis, which could be responsible for its role on callus formation. This study provides novel insights into the role of IGMTs gene-mediated callus formation. Activation of the Indole glucosinolate anabolic pathway is an inducing factor for plant callus initiation. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01409-2.

Genome-wide profiles of DNA damage represent highly accurate predictors of mammalian age.

The identification of novel age-related biomarkers represents an area of intense research interest. Despite multiple studies associating DNA damage with aging, there is a glaring paucity of DNA damage-based biomarkers of age, mainly due to the lack of precise methods for genome-wide surveys of different types of DNA damage. Recently, we developed two techniques for genome-wide mapping of the most prevalent types of DNA damage, single-strand breaks and abasic sites, with nucleotide-level resolution. Herein, we explored the potential of genomic patterns of DNA damage identified by these methods as a source of novel age-related biomarkers using mice as a model system. Strikingly, we found that models based on genomic patterns of either DNA lesion could accurately predict age with higher precision than the commonly used transcriptome analysis. Interestingly, the informative patterns were limited to relatively few genes and the DNA damage levels were positively or negatively correlated with age. These findings show that previously unexplored high-resolution genomic patterns of DNA damage contain useful information that can contribute significantly to both practical applications and basic science.

Evidence for widespread existence of functional novel and non-canonical human transcripts.

BACKGROUND: Fraction of functional sequence in the human genome remains a key unresolved question in Biology and the subject of vigorous debate. While a plethora of studies have connected a significant fraction of human DNA to various biochemical processes, the classical definition of function requires evidence of effects on cellular or organismal fitness that such studies do not provide. Although multiple high-throughput reverse genetics screens have been developed to address this issue, they are limited to annotated genomic elements and suffer from non-specific effects, arguing for a strong need to develop additional functional genomics approaches. RESULTS: In this work, we established a high-throughput lentivirus-based insertional mutagenesis strategy as a forward genetics screen tool in aneuploid cells. Application of this approach to human cell lines in multiple phenotypic screens suggested the presence of many yet uncharacterized functional elements in the human genome, represented at least in part by novel exons of known and novel genes. The novel transcripts containing these exons can be massively, up to thousands-fold, induced by specific stresses, and at least some can represent bi-cistronic protein-coding mRNAs. CONCLUSIONS: Altogether, these results argue that many unannotated and non-canonical human transcripts, including those that appear as aberrant splice products, have biological relevance under specific biological conditions.

Genome-Wide Profiling of Endogenous Single-Stranded DNA Using the SSiNGLe-P1 Method.

Endogenous single-stranded DNA (essDNA) can form in a mammalian genome as the result of a variety of molecular processes and can both play important roles inside the cell as well as have detrimental consequences to genome integrity, much of which remains to be fully understood. Here, we established the SSiNGLe-P1 approach based on limited digestion by P1 endonuclease for high-throughput genome-wide identification of essDNA regions. We applied this method to profile essDNA in both human mitochondrial and nuclear genomes. In the mitochondrial genome, the profiles of essDNA provide new evidence to support the strand-displacement model of mitochondrial DNA replication. In the nuclear genome, essDNA regions were found to be enriched in certain types of functional genomic elements, particularly, the origins of DNA replication, R-loops, and to a lesser degree, in promoters. Furthermore, interestingly, many of the essDNA regions identified by SSiNGLe-P1 have not been annotated and thus could represent yet unknown functional elements.

Lessons from discovery of true ADAR RNA editing sites in a human cell line.

BACKGROUND: Conversion or editing of adenosine (A) into inosine (I) catalyzed by specialized cellular enzymes represents one of the most common post-transcriptional RNA modifications with emerging connection to disease. A-to-I conversions can happen at specific sites and lead to increase in proteome diversity and changes in RNA stability, splicing, and regulation. Such sites can be detected as adenine-to-guanine sequence changes by next-generation RNA sequencing which resulted in millions reported sites from multiple genome-wide surveys. Nonetheless, the lack of extensive independent validation in such endeavors, which is critical considering the relatively high error rate of next-generation sequencing, leads to lingering questions about the validity of the current compendiums of the editing sites and conclusions based on them. RESULTS: Strikingly, we found that the current analytical methods suffer from very high false positive rates and that a significant fraction of sites in the public databases cannot be validated. In this work, we present potential solutions to these problems and provide a comprehensive and extensively validated list of A-to-I editing sites in a human cancer cell line. Our findings demonstrate that most of true A-to-I editing sites in a human cancer cell line are located in the non-coding transcripts, the so-called RNA 'dark matter'. On the other hand, many ADAR editing events occurring in exons of human protein-coding mRNAs, including those that can recode the transcriptome, represent false positives and need to be interpreted with caution. Nonetheless, yet undiscovered authentic ADAR sites that increase the diversity of human proteome exist and warrant further identification. CONCLUSIONS: Accurate identification of human ADAR sites remains a challenging problem, particularly for the sites in exons of protein-coding mRNAs. As a result, genome-wide surveys of ADAR editome must still be accompanied by extensive Sanger validation efforts. However, given the vast number of unknown human ADAR sites, there is a need for further developments of the analytical techniques, potentially those that are based on deep learning solutions, in order to provide a quick and reliable identification of the editome in any sample.

Evidence for Existence of Multiple Functional Human Small RNAs Derived from Transcripts of Protein-Coding Genes.

The human genome encodes a multitude of different noncoding transcripts that have been traditionally separated on the basis of their lengths into long (>200 nt) or small (<200 nt) noncoding RNAs. The functions, mechanisms of action, and biological relevance of the vast majority of both long and short noncoding transcripts remain unknown. However, according to the functional understanding of the known classes of long and small noncoding RNAs (sncRNAs) that have been shown to play crucial roles in multiple biological processes, it is generally assumed that many unannotated long and small transcripts participate in important cellular functions as well. Nevertheless, direct evidence of functionality is lacking for most noncoding transcripts, especially for sncRNAs that are often dismissed as stable degradation products of longer RNAs. Here, we developed a high-throughput assay to test the functionality of sncRNAs by overexpressing them in human cells. Surprisingly, we found that a significant fraction (>40%) of unannotated sncRNAs appear to have biological relevance. Furthermore, contrary to the expectation, the potentially functional transcripts are not highly abundant and can be derived from protein-coding mRNAs. These results strongly suggest that the small noncoding transcriptome can harbor multiple functional transcripts that warrant future studies.

Complex genomic patterns of abasic sites in mammalian DNA revealed by a high-resolution SSiNGLe-AP method.

DNA damage plays a critical role in biology and diseases; however, how different types of DNA lesions affect cellular functions is far from clear mostly due to the paucity of high-resolution methods that can map their locations in complex genomes, such as those of mammals. Here, we present the development and validation of SSiNGLe-AP method, which can map a common type of DNA damage, abasic (AP) sites, in a genome-wide and high-resolution manner. We apply this method to six different tissues of mice with different ages and human cancer cell lines. We find a nonrandom distribution of AP sites in the mammalian genome that exhibits dynamic enrichment at specific genomic locations, including single-nucleotide hotspots, and is significantly influenced by gene expression, age and tissue type in particular. Overall, these results suggest that we are only starting to understand the true complexities in the genomic patterns of DNA damage.

Graphene Oxide Exhibits Antifungal Activity against Bipolaris sorokiniana In Vitro and In Vivo.

The antimicrobial properties of graphene in vitro have been widely reported. However, compared to research performed on graphene's antibacterial properties, there have been relatively few studies assessing graphene's antifungal properties. In particular, evaluating graphene's pathogenic effects on host plants in vivo, which is critical to using graphene in disease control, has rarely been performed. In this study, the fungal pathogen of wheat, barley, and other plants, Bipolaris sorokiniana (B. sorokiniana) and graphene oxide (GO) were selected for materials. A combination of physiological, cytological, and biochemical approaches was used to explore how GO affects the growth and pathogenicity of B. sorokiniana. The mycelial growth and spore germination of B. sorokiniana were both inhibited in a dose-dependent manner by GO treatment. The addition of GO significantly alleviated the infection of pathogenic fungi in host plants. The results of scanning electron microscopy demonstrated that the inhibitory effect of GO on B. sorokiniana was primarily related to the destruction of the cell membrane. Our study confirmed the antifungal effect of graphene in vitro and in vivo, providing an experimental basis for applying graphene in disease resistance, which is of great significance for agricultural and forestry production.

Hotspots of single-strand DNA "breakome" are enriched at transcriptional start sites of genes.

Single-strand breaks (SSBs) represent one of the most common types of DNA damage, yet not much is known about the genome landscapes of this type of DNA lesions in mammalian cells. Here, we found that SSBs are more likely to occur in certain positions of the human genome-SSB hotspots-in different cells of the same cell type and in different cell types. We hypothesize that the hotspots are likely to represent biologically relevant breaks. Furthermore, we found that the hotspots had a prominent tendency to be enriched in the immediate vicinity of transcriptional start sites (TSSs). We show that these hotspots are not likely to represent technical artifacts or be caused by common mechanisms previously found to cause DNA cleavage at promoters, such as apoptotic DNA fragmentation or topoisomerase type II (TOP2) activity. Therefore, such TSS-associated hotspots could potentially be generated using a novel mechanism that could involve preferential cleavage at cytosines, and their existence is consistent with recent studies suggesting a complex relationship between DNA damage and regulation of gene expression.

Methods to Analyze the Non-Coding RNA Interactome-Recent Advances and Challenges.

Most of the human genome is transcribed to generate a multitude of non-coding RNAs. However, while these transcripts have generated an immense amount of scientific interest, their biological function remains a subject of an intense debate. Understanding mechanisms of action of non-coding RNAs is a key to addressing the issue of biological relevance of these transcripts. Based on some well-understood non-coding RNAs that function inside the cell by interacting with other molecules, it is generally believed many other non-coding transcripts could also function in a similar fashion. Therefore, development of methods that can map RNA interactome is the key to understanding functionality of the extensive cellular non-coding transcriptome. Here, we review the vast progress that has been made in the past decade in technologies that can map RNA interactions with different sites in DNA, proteins or other RNA molecules; the general approaches used to validate the existence of novel interactions; and the challenges posed by interpreting the data obtained using the interactome mapping methods.

The Effects of Graphene-Family Nanomaterials on Plant Growth: A Review.

Numerous reports of graphene-family nanomaterials (GFNs) promoting plant growth have opened up a wide range of promising potential applications in agroforestry. However, several toxicity studies have raised growing concerns about the biosafety of GFNs. Although these studies have provided clues about the role of GFNs from different perspectives (such as plant physiology, biochemistry, cytology, and molecular biology), the mechanisms by which GFNs affect plant growth remain poorly understood. In particular, a systematic collection of data regarding differentially expressed genes in response to GFN treatment has not been conducted. We summarize here the fate and biological effects of GFNs in plants. We propose that soil environments may be conducive to the positive effects of GFNs but may be detrimental to the absorption of GFNs. Alterations in plant physiology, biochemistry, cytological structure, and gene expression in response to GFN treatment are discussed. Coincidentally, many changes from the morphological to biochemical scales, which are caused by GFNs treatment, such as affecting root growth, disrupting cell membrane structure, and altering antioxidant systems and hormone concentrations, can all be mapped to gene expression level. This review provides a comprehensive understanding of the effects of GFNs on plant growth to promote their safe and efficient use.

Effects of graphene on morphology, microstructure and transcriptomic profiling of Pinus tabuliformis Carr. roots.

Graphene has shown great potential for improving growth of many plants, but its effect on woody plants remains essentially unstudied. In this work, Pinus tabuliformis Carr. bare-rooted seedlings grown outdoors in pots were irrigated with a graphene solution over a concentration range of 0-50 mg/L for six months. Graphene was found to stimulate root growth, with a maximal effect at 25 mg/L. We then investigated root microstructure and carried out transcript profiling of root materials treated with 0 and 25 mg/L graphene. Graphene treatment resulted in plasma-wall separation and destruction of membrane integrity in root cells. More than 50 thousand of differentially expressed genes (DEGs) were obtained by RNA sequencing, among which 6477 could be annotated using other plant databases. The GO enrichment analysis and KEGG pathway analysis of the annotated DEGs indicated that abiotic stress responses, which resemble salt stress, were induced by graphene treatment in roots, while responses to biotic stimuli were inhibited. Numerous metabolic processes and hormone signal transduction pathways were altered by the treatment. The growth promotion effects of graphene may be mediated by encouraging proline synthesis, and suppression of the expression of the auxin response gene SMALL AUXIN UP-REGULATED RNA 41 (SAUR41), PYL genes which encode ABA receptors, and GSK3 homologs.

Graphene oxide exhibited positive effects on the growth of Aloe vera L.

There is increasing evidence for graphene associated plant growth promotion, however, the chronic effects of soil-applied graphene remain largely unexplored. The present study investigated the morphological, physiological and biochemical responses of graphene oxide (GO) on Aloe vera L. over the concentration range of 0-100 mg/L for four months. Our results demonstrated that GO, with the best efficiency at 50 mg/L, could enhance the photosynthetic capacity of leaves, increase the yield and morphological characters of root and leaf, improve the nutrient (protein and amino acid) contents of leaf, without reducing the content of the main bioactive compound aloin. Compared with leaves, the effect of GO on root growth was more obvious. Although the electrolyte leakage and MDA content were raised at high concentrations, GO treatment did not increase the root antioxidant enzymes activity or decrease the root vigor, which excluding typical stress response. Furthermore, injection experiments showed that the GO in vivo did not change the plant growth state obviously. Taken together, our study revealed the role of GO in promoting Aloe vera growth by stimulating root growth and photosynthesis, which would provide theory basis for GO application in agriculture and forestry. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-00979-3.

Very long intergenic non-coding (vlinc) RNAs directly regulate multiple genes in cis and trans.

BACKGROUND: The majority of the human genome is transcribed in the form of long non-coding (lnc) RNAs. While these transcripts have attracted considerable interest, their molecular mechanisms of function and biological significance remain controversial. One of the main reasons behind this lies in the significant challenges posed by lncRNAs requiring the development of novel methods and concepts to unravel their functionality. Existing methods often lack cross-validation and independent confirmation by different methodologies and therefore leave significant ambiguity as to the authenticity of the outcomes. Nonetheless, despite all the caveats, it appears that lncRNAs may function, at least in part, by regulating other genes via chromatin interactions. Therefore, the function of a lncRNA could be inferred from the function of genes it regulates. In this work, we present a genome-wide functional annotation strategy for lncRNAs based on identification of their regulatory networks via the integration of three distinct types of approaches: co-expression analysis, mapping of lncRNA-chromatin interactions, and assaying molecular effects of lncRNA knockdowns obtained using an inducible and highly specific CRISPR/Cas13 system. RESULTS: We applied the strategy to annotate 407 very long intergenic non-coding (vlinc) RNAs belonging to a novel widespread subclass of lncRNAs. We show that vlincRNAs indeed appear to regulate multiple genes encoding proteins predominantly involved in RNA- and development-related functions, cell cycle, and cellular adhesion via a mechanism involving proximity between vlincRNAs and their targets in the nucleus. A typical vlincRNAs can be both a positive and negative regulator and regulate multiple genes both in trans and cis. Finally, we show vlincRNAs and their regulatory networks potentially represent novel components of DNA damage response and are functionally important for the ability of cancer cells to survive genotoxic stress. CONCLUSIONS: This study provides strong evidence for the regulatory role of the vlincRNA class of lncRNAs and a potentially important role played by these transcripts in the hidden layer of RNA-based regulation in complex biological systems.

Hovlinc is a recently evolved class of ribozyme found in human lncRNA.

Although naturally occurring catalytic RNA molecules-ribozymes-have attracted a great deal of research interest, very few have been identified in humans. Here, we developed a genome-wide approach to discovering self-cleaving ribozymes and identified a naturally occurring ribozyme in humans. The secondary structure and biochemical properties of this ribozyme indicate that it belongs to an unidentified class of small, self-cleaving ribozymes. The sequence of the ribozyme exhibits a clear evolutionary path, from its appearance between ~130 and ~65 million years ago (Ma), to acquiring self-cleavage activity very recently, ~13-10 Ma, in the common ancestors of humans, chimpanzees and gorillas. The ribozyme appears to be functional in vivo and is embedded within a long noncoding RNA belonging to a class of very long intergenic noncoding RNAs. The presence of a catalytic RNA enzyme in lncRNA creates the possibility that these transcripts could function by carrying catalytic RNA domains.

A CRISPR/Cas13-based approach demonstrates biological relevance of vlinc class of long non-coding RNAs in anticancer drug response.

Long non-coding (lnc) RNAs represent a fascinating class of transcripts that remains highly controversial mainly due to ambiguity surrounding overall biological relevance of these RNAs. Multitude of reverse genetics studies showing functionality of lncRNAs are unfortunately based on assays that are either plagued by non-specific effects and/or cannot unambiguously assign observed phenotypes to the transcript per se. Here, we show application of the novel CRISPR/Cas13 RNA knockdown system that has superior specificity compared to other transcript-targeting knockdown methods like RNAi. We applied this method to a novel widespread subclass of nuclear lncRNAs - very long intergenic non-coding (vlinc) RNAs - in a high-throughput phenotypic assay based on survival challenge in response to anticancer drug treatments. We used multiple layers of controls including mismatch control for each targeting gRNA to ensure uncovering true phenotype-transcript relationships. We found evidence supporting importance for cellular survival for up to 60% of the tested protein-coding mRNAs and, importantly, 64% of vlincRNAs. Overall, this study demonstrates utility of CRISPR/Cas13 as a highly sensitive and specific tool for reverse genetics study of both protein-coding genes and lncRNAs. Furthermore, importantly, this approach provides evidence supporting biological significance of the latter transcripts in anticancer drug response.

Novel approach reveals genomic landscapes of single-strand DNA breaks with nucleotide resolution in human cells.

Single-strand breaks (SSBs) represent the major form of DNA damage, yet techniques to map these lesions genome-wide with nucleotide-level precision are limited. Here, we present a method, termed SSiNGLe, and demonstrate its utility to explore the distribution and dynamic changes in genome-wide SSBs in response to different biological and environmental stimuli. We validate SSiNGLe using two very distinct sequencing techniques and apply it to derive global profiles of SSBs in different biological states. Strikingly, we show that patterns of SSBs in the genome are non-random, specific to different biological states, enriched in regulatory elements, exons, introns, specific types of repeats and exhibit differential preference for the template strand between exons and introns. Furthermore, we show that breaks likely contribute to naturally occurring sequence variants. Finally, we demonstrate strong links between SSB patterns and age. Overall, SSiNGLe provides access to unexplored realms of cellular biology, not obtainable with current approaches.