Non-coding Natural Antisense Transcripts Research Articles

Comprehensive investigation of long non-coding RNAs in an endophytic fungus Calcarisporium arbuscula NRRL 3705.

Long non-coding RNAs (lncRNAs) play an important role in eukaryotic cells. However, there is no report of lncRNAs in endophytic fungi Calcarisporium arbuscula. Here, in Calcarisporium arbuscula NRRL 3705, an endophytic fungus predominantly producing mycotoxins aurovertins, the genome-wide identification of lncRNAs was carried out based on RNA-Seq. Totally, 1332 lncRNAs were identified, including 1082 long intergenic noncoding RNAs, 64 long intronic noncoding RNAs and 186 long noncoding natural antisense transcripts. The average length of lncRNA and mRNA were 254 and 1102bp, respectively. LncRNAs were shorter, with fewer exons and lower expression levels. Moreover, there were 39 up-regulated lncRNAs and 10 down-regulated lncRNAs in the ΔaurA mutant, which lacks the aurovertin biosynthetic enzyme AurA. Interestingly, expression of genes related to the metabolism of linoleic acid and methane were significantly down regulated in the ΔaurA mutant. This study enriches the endophytic fungal lncRNA database and provide a basis for further research.

Genome-wide identification of antisense lncRNAs and their association with susceptibility to Flavobacterium psychrophilum in rainbow trout.

Eukaryotic genomes encode long noncoding natural antisense transcripts (lncNATs) that have been increasingly recognized as regulatory members of gene expression. Recently, we identified a few antisense transcripts correlating in expression with immune-related genes. However, a systematic genome-wide analysis of lncNATs in rainbow trout is lacking. This study used 134 RNA-Seq datasets from five different projects to identify antisense transcripts. A total of 13,503 lncNATs were identified genome-wide. About 75% of lncNATs showed multiple exons compared to 36.5% of the intergenic lncRNAs. RNA-Seq datasets from resistant, control, and susceptible rainbow trout genetic lines with significant differences in survival rate following Flavobacterium psychrophilum (Fp) infection were analyzed to investigate the potential role of the lncNATs during infection. Twenty-four pairwise comparisons between the different genetic lines, infectious status, and time points revealed 581 differentially expressed (DE) lncNATs and 179 differentially used exons (DUEs). Most of the DE lncNATs strongly and positively correlated in expression with their corresponding sense transcripts across 24 RNA-Seq datasets. LncNATs complementary to genes related to immunity, muscle contraction, proteolysis, and iron/heme metabolism were DE following infection. LncNATs complementary to hemolysis-related genes were DE in the resistant fish compared to susceptible fish on day 5 post-infection, suggesting enhanced clearance of free hemoglobin (Hb) and heme and increased erythropoiesis. LncNATs complementary to hepcidin, a master negative regulator of the plasma iron concentration, were the most downregulated lncNATs on day 5 of bacterial infection in the resistant fish. Ninety-four DE lncNAT, including five complementary to hepcidin, are located within 26 QTL regions previously identified in association with bacterial cold water disease (BCWD) in rainbow trout. Collectively, lncNATs are involved in the molecular architecture of fish immunity and should be further investigated for potential applications in genomic selection and genetic manipulation in aquaculture.

Transcriptome analysis reveals long non-coding natural antisense transcripts involved in muscle development in fetal goat (Capra hircus).

Non-coding RNAs have been shown to play vital roles in muscle development. However, the biological roles of long non-coding natural antisense transcripts, antisense lncRNAs (ASlncRNAs), are largely unknown in embryonic muscle development. Here, we identified a total of 466 ASlncRNAs in the longissimus dorsi muscle. And 48 differentially expressed ASlncRNAs were identified based on pairwise comparisons (P<0.05), sixteen of which were validated by qPCR. Additionally, 466 ASlncRNAs were predicted to target 335 protein coding genes based on complementary base-pairing. Enrichment analysis suggests that ASlncRNAs may be involved in muscle development by negatively regulating the expression of target genes. Furthermore, 170 ASlncRNAs were identified as potential miRNA precursors, suggesting that these ASlncRNAs may be involved in the regulation of muscle development by producing miRNA precursors. Our results provide a catalog of goat muscle-related ASlncRNAs, and will contribute to a fuller understanding of the roles of ASlncRNAs in muscle development.

Making sense of the natural antisense transcript puzzle.

In plants, thousands of genes are associated with antisense transcription, which often produces noncoding RNAs. Although widespread, sense-antisense pairs have been implicated in a limited variety of functions in plants and are often thought to form extensive dsRNA stretches triggering gene silencing. In this opinion, we show that evidence does not support gene silencing as a major role for antisense transcription. In fact, it is more likely that antisense transcripts play diverse functions in gene regulation. We propose a general framework for the initial functional dissection of antisense transcripts, suggesting testable hypotheses relying on an experiment-based decision tree. By moving beyond the gene silencing paradigm, we argue that a broad and diverse role for natural antisense transcription will emerge.

Antisense RNAs Influence Promoter Usage of Their Counterpart Sense Genes in Cancer.

Multiple noncoding natural antisense transcripts (ncNAT) are known to modulate key biological events such as cell growth or differentiation. However, the actual impact of ncNATs on cancer progression remains largely unknown. In this study, we identified a complete list of differentially expressed ncNATs in hepatocellular carcinoma. Among them, a previously undescribed ncNAT HNF4A-AS1L suppressed cancer cell growth by regulating its sense gene HNF4A, a well-known cancer driver, through a promoter-specific mechanism. HNF4A-AS1L selectively activated the HNF4A P1 promoter via HNF1A, which upregulated expression of tumor suppressor P1-driven isoforms, while having no effect on the oncogenic P2 promoter. RNA-seq data from 23 tissue and cancer types identified approximately 100 ncNATs whose expression correlated specifically with the activity of one promoter of their associated sense gene. Silencing of two of these ncNATs ENSG00000259357 and ENSG00000255031 (antisense to CERS2 and CHKA, respectively) altered the promoter usage of CERS2 and CHKA. Altogether, these results demonstrate that promoter-specific regulation is a mechanism used by ncNATs for context-specific control of alternative isoform expression of their counterpart sense genes. SIGNIFICANCE: This study characterizes a previously unexplored role of ncNATs in regulation of isoform expression of associated sense genes, highlighting a mechanism of alternative promoter usage in cancer.

Non-coding Natural Antisense Transcripts: Analysis and Application

Non-coding natural antisense transcripts (ncNATs) are regulatory RNA molecules that are overlapping with as well as complementary to other transcripts. These transcripts are implicated in a broad variety of biological and pathological processes, including tumorigenesis and oncogenic progression. With this complex field still in its infancy, annotations, expression profiling and functional characterisations of ncNATs are far less comprehensive than those for protein-coding genes, pointing out substantial gaps in the analysis and characterisation of these regulatory transcripts. In this review, we discuss ncNATs from an analysis perspective, in particular regarding the use of high-throughput sequencing strategies, such as RNA-sequencing, and summarize the unique challenges of investigating the antisense transcriptome. Finally, we elaborate on their potential as biomarkers and future targets for treatment, focusing on cancer.

Identification of long noncoding natural antisense transcripts (lncNATs) correlated with drought stress response in wild rice (Oryza nivara)

BackgroundWild rice, including Oryza nivara and Oryza rufipogon, which are considered as the ancestors of Asian cultivated rice (Oryza sativa), possess high genetic diversity and serve as a crucial resource for breeding novel cultivars of cultivated rice. Although rice domestication related traits, such as seed shattering and plant architecture, have been intensively studied at the phenotypic and genomic levels, further investigation is needed to understand the molecular basis of phenotypic differences between cultivated and wild rice. Drought stress is one of the most severe abiotic stresses affecting rice growth and production. Adaptation to drought stress involves a cascade of genes and regulatory factors that form complex networks. O. nivara inhabits swampy areas with a seasonally dry climate, which is an ideal material to discover drought tolerance alleles. Long noncoding natural antisense transcripts (lncNATs), a class of long noncoding RNAs (lncRNAs), regulate the corresponding sense transcripts and play an important role in plant growth and development. However, the contribution of lncNATs to drought stress response in wild rice remains largely unknown.ResultsHere, we conducted strand-specific RNA sequencing (ssRNA-seq) analysis of Nipponbare (O. sativa) and two O. nivara accessions (BJ89 and BJ278) to determine the role of lncNATs in drought stress response in wild rice. A total of 1246 lncRNAs were identified, including 1091 coding–noncoding NAT pairs, of which 50 were expressed only in Nipponbare, and 77 were expressed only in BJ89 and/or BJ278. Of the 1091 coding–noncoding NAT pairs, 240 were differentially expressed between control and drought stress conditions. Among these 240 NAT pairs, 12 were detected only in Nipponbare, and 187 were detected uniquely in O. nivara. Furthermore, 10 of the 240 coding–noncoding NAT pairs were correlated with genes enriched in stress responsive GO terms; among these, nine pairs were uniquely found in O. nivara, and one pair was shared between O. nivara and Nipponbare.ConclusionWe identified lncNATs associated with drought stress response in cultivated rice and O. nivara. These results will improve our understanding of the function of lncNATs in drought tolerance and accelerate rice breeding.

Mutation of a major CG methylase alters genome-wide lncRNA expression in rice

Plant long non-coding RNAs (lncRNAs) function in diverse biological processes, and lncRNA expression is under epigenetic regulation, including by cytosine DNA methylation. However, it remains unclear whether 5-methylcytosine (5mC) plays a similar role in different sequence contexts (CG, CHG, and CHH). In this study, we characterized and compared the profiles of genome-wide lncRNA profiles (including long intergenic non-coding RNAs [lincRNAs] and long noncoding natural antisense transcripts [lncNATs]) of a null mutant of the rice DNA methyltransferase 1, OsMET1-2 (designated OsMET1-2−/−) and its isogenic wild type (OsMET1-2+/+). The En/Spm transposable element (TE) family, which was heavily methylated in OsMET1-2+/+, was transcriptionally de-repressed in OsMET1-2−/− due to genome-wide erasure of CG methylation, and this led to abundant production of specific lncRNAs. In addition, RdDM-mediated CHH hypermethylation was increased in the 5′-upstream genomic regions of lncRNAs in OsMET1-2−/−. The positive correlation between the expression of lincRNAs and that of their proximal protein-coding genes was also analyzed. Our study shows that CG methylation negatively regulates the TE-related expression of lncRNA and demonstrates that CHH methylation is also involved in the regulation of lncRNA expression.

Time dependent differential regulation of a novel long non-coding natural antisense RNA during long-term memory formation

Long natural antisense transcripts (NATs) have been demonstrated in significant numbers in a variety of eukaryotic organisms. They are particularly prevalent in the nervous system suggesting their importance in neural functions. However, the precise physiological roles of the overwhelming majority of long NATs remain unclear. Here we report on the characterization of a novel molluscan nitric oxide synthase (NOS)-related long non-coding NAT (Lym-NOS1AS). This NAT is spliced and polyadenylated and is transcribed from the non-template strand of the Lym-NOS1 gene. We demonstrate that the Lym-NOS1AS is co-expressed with the sense Lym-NOS1 mRNA in a key neuron of memory network. Also, we report that the Lym-NOS1AS is temporally and spatially regulated by one-trial conditioning leading to long term memory (LTM) formation. Specifically, in the cerebral, but not in the buccal ganglia, the temporal pattern of changes in Lym-NOS1AS expression after training correlates with the alteration of memory lapse and non-lapse periods. Our data suggest that the Lym-NOS1AS plays a role in the consolidation of nitric oxide-dependent LTM.

Global identification of a marine diatom long noncoding natural antisense transcripts (NATs) and their response to phosphate fluctuations

Often ignored and regarded as mere transcriptional noise, long noncoding RNAs (lncRNAs) are starting to be considered key regulators of gene expression across the Eukarya domain of life. In the model diatom Phaeodactylum tricornutum, we have previously reported the occurrence of 1,510 intergenic lncRNAs (lincRNAs), many of which displaying specific patterns of expression under phosphate fluctuation (Pi). Using strand-specific RNA-sequencing data we now expand the repertoire of P. tricornutum lncRNAs by identifying 2,628 novel natural antisense transcripts (NATs) that cover 21.5% of the annotated genomic loci. We found that NAT expression is tightly regulated by phosphate depletion and other naturally occurring environmental stresses. Furthermore, we identified 121 phosphate stress responsive NAT-mRNA pairs, the great majority of which showing a positive correlation (concordant pairs) and a small fraction with negative correlation (discordant pairs). Taken together our results show that NATs are highly abundant transcripts in P. tricornutum and that their expression is under tight regulation by nutrient and environmental stresses. Furthermore, our results suggest that in P. tricornutum Pi stress response NAT pairs predominantly regulate positively the expression of their cognate sense genes, the latter being involved in several biological processes underlying the control of cellular homeostasis under stress.

Identification and characterization of pineapple leaf lncRNAs in crassulacean acid metabolism (CAM) photosynthesis pathway

Long noncoding RNAs (lncRNAs) have been identified in many mammals and plants and are known to play crucial roles in multiple biological processes. Pineapple is an important tropical fruit and a good model for studying the plant evolutionary adaptation to the dry environment and the crassulacean acid metabolism (CAM) photosynthesis strategy; however, the lncRNAs involved in CAM pathway remain poorly characterized. Here, we analyzed the available RNA-seq data sets derived from 26 pineapple leaf samples at 13 time points and identified 2,888 leaf lncRNAs, including 2,046 long intergenic noncoding RNAs (lincRNAs) and 842 long noncoding natural antisense transcripts (lncNATs). Pineapple leaf lncRNAs are expressed in a highly tissue-specific manner. Co-expression analysis of leaf lncRNA and mRNA revealed that leaf lncRNAs are preferentially associated with photosynthesis genes. We further identified leaf lncRNAs that potentially function as competing endogenous RNAs (ceRNAs) of two CAM photosynthesis pathway genes, PPCK and PEPC, and revealed their diurnal expression pattern in leaves. Moreover, we found that 48% of lncRNAs exhibit diurnal expression patterns in leaves, suggesting their important roles in CAM. This study conducted a comprehensive genome-wide analysis of leaf lncRNAs and identified their role in gene expression regulation of the CAM photosynthesis pathway in pineapple.

Bioinformatics Approaches to Studying Plant Long Noncoding RNAs (lncRNAs): Identification and Functional Interpretation of lncRNAs from RNA-Seq Data Sets.

Long noncoding RNAs (lncRNAs) play important roles in regulating various biological processes including growth and stress responses in plants. RNA-seq data sets provide a good resource to exploring the noncoding transcriptome and studying their comprehensive interactions with the coding transcriptome. Here, we describe computational procedures for studying plant lncRNAs including long intergenic noncoding RNAs (lincRNAs) and long noncoding natural antisense transcripts (lncNATs). Bioinformatics tools for transcriptome assembly, lncRNA identification, and functional interpretations are included. Finally, we also introduce PLncDB, a user-friendly database that provides comprehensive information of plant lncRNAs for researchers to compare their own data sets to those in public database.

Re-analysis of long non-coding RNAs and prediction of circRNAs reveal their novel roles in susceptible tomato following TYLCV infection

BackgroundLong Noncoding-RNAs (LncRNAs) are known to be involved in some biological processes, but their roles in plant-virus interactions remain largely unexplored. While circular RNAs (circRNAs) have been studied in animals, there has yet to be extensive research on them in a plant system, especially in tomato-tomato yellow leaf curl virus (TYLCV) interaction.ResultsIn this study, RNA transcripts from the susceptible tomato line JS-CT-9210 either infected with TYLCV or untreated, were sequenced in a pair-end strand-specific manner using ribo-zero rRNA removal library method. A total of 2056 lncRNAs including 1767 long intergenic non-coding RNA (lincRNAs) and 289 long non-coding natural antisense transcripts (lncNATs) were obtained. The expression patterns in lncRNAs were similar in susceptible tomato plants between control check (CK) and TYLCV infected samples. Our analysis suggested that lncRNAs likely played a role in a variety of functions, including plant hormone signaling, protein processing in the endoplasmic reticulum, RNA transport, ribosome function, photosynthesis, glulathione metabolism, and plant-pathogen interactions. Using virus-induced gene silencing (VIGS) analysis, we found that reduced expression of the lncRNA S-slylnc0957 resulted in enhanced resistance to TYLCV in susceptible tomato plants. Moreover, we identified 184 circRNAs candidates using the CircRNA Identifier (CIRI) software, of which 32 circRNAs were specifically expressed in untreated samples and 83 circRNAs in TYLCV samples. Approximately 62% of these circRNAs were derived from exons. We validated the circRNAs by both PCR and Sanger sequencing using divergent primers, and found that most of circRNAs were derived from the exons of protein coding genes. The silencing of these circRNAs parent genes resulted in decreased TYLCV virus accumulation.ConclusionIn this study, we identified novel lncRNAs and circRNAs using bioinformatic approaches and showed that these RNAs function as negative regulators of TYLCV infection. Moreover, the expression patterns of lncRNAs in susceptible tomato plants were different from that of resistant tomato plants, while exonic circRNAs expression positively associated with their respective protein coding genes. This work provides a foundation for elaborating the novel roles of lncRNAs and circRNAs in susceptible tomatoes following TYLCV infection.

Transcriptome-wide analysis of natural antisense transcripts shows their potential role in breast cancer

Non-coding RNAs (ncRNA) represent 1/5 of the mammalian transcript number, and 90% of the genome length is transcribed. Many ncRNAs play a role in cancer. Among them, non-coding natural antisense transcripts (ncNAT) are RNA sequences that are complementary and overlapping to those of either protein-coding (PCT) or non-coding transcripts. Several ncNATs were described as regulating protein coding gene expression on the same loci, and they are expected to act more frequently in cis compared to other ncRNAs that commonly function in trans. In this work, 22 breast cancers expressing estrogen receptors and their paired adjacent non-malignant tissues were analyzed by strand-specific RNA sequencing. To highlight ncNATs potentially playing a role in protein coding gene regulations that occur in breast cancer, three different data analysis methods were used: differential expression analysis of ncNATs between tumor and non-malignant tissues, differential correlation analysis of paired ncNAT/PCT between tumor and non-malignant tissues, and ncNAT/PCT read count ratio variation between tumor and non-malignant tissues. Each of these methods yielded lists of ncNAT/PCT pairs that were enriched in survival-associated genes. This work highlights ncNAT lists that display potential to affect the expression of protein-coding genes involved in breast cancer pathology.

A novel long non-coding natural antisense RNA is a negative regulator of Nos1 gene expression.

Long non-coding natural antisense transcripts (NATs) are widespread in eukaryotic species. Although recent studies indicate that long NATs are engaged in the regulation of gene expression, the precise functional roles of the vast majority of them are unknown. Here we report that a long NAT (Mm-antiNos1 RNA) complementary to mRNA encoding the neuronal isoform of nitric oxide synthase (Nos1) is expressed in the mouse brain and is transcribed from the non-template strand of the Nos1 locus. Nos1 produces nitric oxide (NO), a major signaling molecule in the CNS implicated in many important functions including neuronal differentiation and memory formation. We show that the newly discovered NAT negatively regulates Nos1 gene expression. Moreover, our quantitative studies of the temporal expression profiles of Mm-antiNos1 RNA in the mouse brain during embryonic development and postnatal life indicate that it may be involved in the regulation of NO-dependent neurogenesis.

The frequency natural antisense transcript first promotes, then represses, frequency gene expression via facultative heterochromatin

The circadian clock is controlled by a network of interconnected feedback loops that require histone modifications and chromatin remodeling. Long noncoding natural antisense transcripts (NATs) originate from Period in mammals and frequency (frq) in Neurospora. To understand the role of NATs in the clock, we put the frq antisense transcript qrf (frq spelled backwards) under the control of an inducible promoter. Replacing the endogenous qrf promoter altered heterochromatin formation and DNA methylation at frq. In addition, constitutive, low-level induction of qrf caused a dramatic effect on the endogenous rhythm and elevated circadian output. Surprisingly, even though qrf is needed for heterochromatic silencing, induction of qrf initially promoted frq gene expression by creating a more permissible local chromatin environment. The observation that antisense expression can initially promote sense gene expression before silencing via heterochromatin formation at convergent loci is also found when a NAT to hygromycin resistance gene is driven off the endogenous vivid (vvd) promoter in the Δvvd strain. Facultative heterochromatin silencing at frq functions in a parallel pathway to previously characterized VVD-dependent silencing and is needed to establish the appropriate circadian phase. Thus, repression via dicer-independent siRNA-mediated facultative heterochromatin is largely independent of, and occurs alongside, other feedback processes.

Genome-wide identification of long noncoding natural antisense transcripts and their responses to light in Arabidopsis

Recent research on long noncoding RNAs (lncRNAs) has expanded our understanding of gene transcription regulation and the generation of cellular complexity. Depending on their genomic origins, lncRNAs can be transcribed from intergenic or intragenic regions or from introns of protein-coding genes. We have recently reported more than 6000 intergenic lncRNAs in Arabidopsis. Here, we systematically identified long noncoding natural antisense transcripts (lncNATs), defined as lncRNAs transcribed from the opposite DNA strand of coding or noncoding genes. We found a total of 37,238 sense–antisense transcript pairs and 70% of annotated mRNAs to be associated with antisense transcripts in Arabidopsis. These lncNATs could be reproducibly detected by different technical platforms, including strand-specific tiling arrays, Agilent custom expression arrays, strand-specific RNA-seq, and qRT-PCR experiments. Moreover, we investigated the expression profiles of sense–antisense pairs in response to light and observed spatial and developmental-specific light effects on 626 concordant and 766 discordant NAT pairs. Genes for a large number of the light-responsive NAT pairs are associated with histone modification peaks, and histone acetylation is dynamically correlated with light-responsive expression changes of NATs.

Long noncoding RNAs responsive to Fusarium oxysporum infection in Arabidopsis thaliana

Short noncoding RNAs have been demonstrated to play important roles in regulation of gene expression and stress responses, but the repertoire and functions of long noncoding RNAs (lncRNAs) remain largely unexplored, particularly in plants. To explore the role of lncRNAs in disease resistance, we used a strand-specific RNA-sequencing approach to identify lncRNAs responsive to Fusarium oxysporum infection in Arabidopsis thaliana. Antisense transcription was found in c. 20% of the annotated A. thaliana genes. Several noncoding natural antisense transcripts responsive to F. oxysporum infection were found in genes implicated in disease defense. While the majority of the novel transcriptionally active regions (TARs) were adjacent to annotated genes and could be an extension of the annotated transcripts, 159 novel intergenic TARs, including 20 F. oxysporum-responsive lncTARs, were identified. Ten F. oxysporum-induced lncTARs were functionally characterized using T-DNA insertion or RNA-interference knockdown lines, and five were demonstrated to be related to disease development. Promoter analysis suggests that some of the F. oxysporum-induced lncTARs are direct targets of transcription factor(s) responsive to pathogen attack. Our results demonstrated that strand-specific RNA sequencing is a powerful tool for uncovering hidden levels of transcriptome and that IncRNAs are important components of the antifungal networks in A. thaliana.