Sense-antisense Gene Pairs Research Articles

Viral Decoys: The Only Two Herpesviruses Infecting Invertebrates Evolved Different Transcriptional Strategies to Deflect Post-Transcriptional Editing.

The highly versatile group of Herpesviruses cause disease in a wide range of hosts. In invertebrates, only two herpesviruses are known: the malacoherpesviruses HaHV-1 and OsHV-1 infecting gastropods and bivalves, respectively. To understand viral transcript architecture and diversity we first reconstructed full-length viral genomes of HaHV-1 infecting Haliotis diversicolor supertexta and OsHV-1 infecting Scapharca broughtonii by DNA-seq. We then used RNA-seq over the time-course of experimental infections to establish viral transcriptional dynamics, followed by PacBio long-read sequencing of full-length transcripts to untangle viral transcript architectures at two selected time points. Despite similarities in genome structure, in the number of genes and in the diverse transcriptomic architectures, we measured a ten-fold higher transcript variability in HaHV-1, with more extended antisense gene transcription. Transcriptional dynamics also appeared different, both in timing and expression trends. Both viruses were heavily affected by post-transcriptional modifications performed by ADAR1 affecting sense-antisense gene pairs forming dsRNAs. However, OsHV-1 concentrated these modifications in a few genomic hotspots, whereas HaHV-1 diluted ADAR1 impact by elongated and polycistronic transcripts distributed over its whole genome. These transcriptional strategies might thus provide alternative potential roles for sense-antisense transcription in viral transcriptomes to evade the host’s immune response in different virus–host combinations.

Computational analysis of sense-antisense chimeric transcripts reveals their potential regulatory features and the landscape of expression in human cells.

Many human genes are transcribed from both strands and produce sense-antisense gene pairs. Sense-antisense (SAS) chimeric transcripts are produced upon the coalescing of exons/introns from both sense and antisense transcripts of the same gene. SAS chimera was first reported in prostate cancer cells. Subsequently, numerous SAS chimeras have been reported in the ChiTaRS-2.1 database. However, the landscape of their expression in human cells and functional aspects are still unknown. We found that longer palindromic sequences are a unique feature of SAS chimeras. Structural analysis indicates that a long hairpin-like structure formed by many consecutive Watson-Crick base pairs appears because of these long palindromic sequences, which possibly play a similar role as double-stranded RNA (dsRNA), interfering with gene expression. RNA–RNA interaction analysis suggested that SAS chimeras could significantly interact with their parental mRNAs, indicating their potential regulatory features. Here, 267 SAS chimeras were mapped in RNA-seq data from 16 healthy human tissues, revealing their expression in normal cells. Evolutionary analysis suggested the positive selection favoring sense-antisense fusions that significantly impacted the evolution of their function and structure. Overall, our study provides detailed insight into the expression landscape of SAS chimeras in human cells and identifies potential regulatory features.

Promoter switching in response to changing environment and elevated expression of protein-coding genes overlapping at their 5\u2019 ends

Despite the number of studies focused on sense-antisense transcription, the key question of whether such organization evolved as a regulator of gene expression or if this is only a byproduct of other regulatory processes has not been elucidated to date. In this study, protein-coding sense-antisense gene pairs were analyzed with a particular focus on pairs overlapping at their 5’ ends. Analyses were performed in 73 human transcription start site libraries. The results of our studies showed that the overlap between genes is not a stable feature and depends on which TSSs are utilized in a given cell type. An analysis of gene expression did not confirm that overlap between genes causes downregulation of their expression. This observation contradicts earlier findings. In addition, we showed that the switch from one promoter to another, leading to genes overlap, may occur in response to changing environment of a cell or tissue. We also demonstrated that in transfected and cancerous cells genes overlap is observed more often in comparison with normal tissues. Moreover, utilization of overlapping promoters depends on particular state of a cell and, at least in some groups of genes, is not merely coincidental.

Investigation of the putative role of antisense transcripts as regulators of sense transcripts by correlation analysis of sense-antisense pairs in colorectal cancers.

Antisense transcription occurs widely more expected than when it was first identified in bacteria in the 1980s. However, the functional relevance of antisense transcripts in transcription remains controversial. Here, we investigated the putative role of antisense transcripts in regulating their corresponding sense transcripts by analyzing changes in correlative relationships between sense-antisense pairs under tumor and normal conditions. A total of 3469 sense-antisense gene pairs (SAGPs) downloaded from BioMart mapped to a list of sense and antisense genes in RNA-seq data derived from 80 paired colorectal cancer (CRC) samples were analyzed. As a result, cancer-related genes were significantly enriched in the significantly correlated SAGPs (SCPs). Differentially expressed genes estimated between normal and tumor conditions were also significantly more enriched in SCPs than in non-SCPs. Interestingly, using differential correlation analysis, we found that tumor samples had a significantly larger density of genes with higher correlation coefficients than normal samples, as verified by various cancer transcriptomes from The Cancer Genome Atlas (TCGA). Moreover, we found that the magnitude of the correlation between SAGPs could distinguish poor prognostic CRCs from good prognostic CRCs, showing that correlation coefficients between the SAGPs of CRCs with a poor prognosis were significantly stronger than CRCs with a good prognosis. Consistent with this finding, the Cox proportion hazards model showed that the survival rates were significantly different between patients with high and low expression of genes in the SCPs. All these results strongly support the idea that antisense transcripts are important regulators of their corresponding sense transcripts.

Candidate Genetic Modifiers for RPGR Retinal Degeneration.

PurposeTo define genetic variants associated with variable severity of X-linked progressive retinal atrophy 1 (XLPRA1) caused by a five-nucleotide deletion in canine RPGR exon ORF15.MethodsA genome-wide association study (GWAS) was performed in XLPRA1 phenotype informative pedigree. Whole genome sequencing (WGS) was used for mutational analysis of genes within the candidate genomic region. Retinas of normal and mutant dogs were used for gene expression, gene structure, and RNA duplex analyses.ResultsGWAS followed by haplotype phasing identified an approximately 4.6 Mb candidate genomic interval on CFA31 containing seven protein-coding genes expressed in retina (ROBO1, ROBO2, RBM11, NRIP1, HSPA13, SAMSN1, and USP25). Furthermore, we identified and characterized two novel lncRNAs, ROBO1-AS and ROBO2-AS, that display overlapping gene organization with axon guidance pathway genes ROBO1 and ROBO2, respectively, producing sense-antisense gene pairs. Notably, ROBO1-AS and ROBO2-AS act in cis to form lncRNA/mRNA duplexes with ROBO1 and ROBO2, respectively, suggesting important roles for these lncRNAs in the ROBO regulatory network. A subsequent WGS identified candidate genes within the genomic region on CFA31 that might be implicated in modifying severity of XLPRA1. This approach led to discovery of genetic variants in ROBO1, ROBO1-AS, ROBO2-AS, and USP25 that are strongly associated with the XLPRA1 moderate phenotype.ConclusionsThe study provides new insights into the genetic basis of phenotypic variation in severity of RPGRorf15-associated retinal degeneration. Our findings suggest an important role for ROBO pathways in disease progression further expanding on our previously reported changes of ROBO1 expression in XLPRA1 retinas.

Abstract 2454: Cancer RNA:DNA interactome analysis and prediction

Abstract Transcriptional R-loops are three-stranded RNA:DNA hybrid structures essential for many normal and pathobiological processes. Previously, we have proposed and validated a quantitative R-loop forming structure (RLFS) model, QmRLFS, and predicted about 660,000 RLFSs in the human genome; most of them are localized in total within 75% of the coding and non-coding genes and gene-flanking regions, transcribed enhancer and pseudogene regions. RLFSs are also commonly co-localized as the singleton or clusters at regulatory sites and disease-associated genomic loci. Recently, it was computationally predicted and experimentally shown that RNA:DNA hybrid/R-loop interactome may play critical roles in mutagenesis, chromosome breaks, genome instability and cancer development. Herein, we conducted a comprehensive comparative analysis between the RLFSs, their clusters, publically available DRIP-seq and DRIPc-seq, and our own experimental datasets. QmRLFS demonstrated high accuracy (82-92%) and unbiased performance with resolution on the nucleotide, gene and genome scales. A preferential co-localization of the RLFSs with promoters, U1 splice sites, gene ends, enhancers and non-B DNA structures, such as G-quadruplexes, provides evidences for the mechanic links between RLFSs and dynamical DNA tertiary structures, transcription initiation and critical regulatory genome signals. We introduced and characterized a class of 25000 paired reverse-forward RLFS clusters highly enriched with non-B DNA structures, which co-localized in the transcribed promoters of sense-antisense gene pairs and enhancer’s flanks. In the cancer genomes, we found the RLFSs that form RNA:DNA hybrids/R-loops and could preferentially link open promoters and transcriptionally active enhancer regions. This suggests new mechanisms for in trans RNA:DNA hybrid-mediated 3D-chromatin loop formation. Finally, we found the RLFS clusters supported by cancer cell RNA:DNA hybrids/R-loop data, which were overlapped with hyper-mutated clusters, kataegis expression signatures. Overall, our study provides a rationale for the prediction, discovery, and characterization of the RLFSs as novel functional structures for an emerging quantitative biology and pathobiology of RNA-DNA interactomes. Citation Format: Vladimir A. Kuznetsov. Cancer RNA:DNA interactome analysis and prediction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2454.

Sense-antisense gene overlap is probably a cause for retaining the few introns in Giardia genome and the implications

BackgroundIt is widely accepted that the last eukaryotic common ancestor and early eukaryotes were intron-rich and intron loss dominated subsequent evolution, thus the presence of only very few introns in some modern eukaryotes must be the consequence of massive loss. But it is striking that few eukaryotes were found to have completely lost introns. Despite extensive research, the causes of massive intron losses remain elusive. Actually the reverse question -- how the few introns can be retained under the evolutionary selection pressure of intron loss -- is equally significant but was rarely studied, except that it was conjectured that the essential functions of some introns prevent their loss. The situation that extremely few (eight) spliceosome-mediated cis-spliced introns present in the relatively simple genome of Giardia lamblia provides an excellent opportunity to explore this question.ResultsOur investigation found three types of distribution patterns of the few introns in the intron-containing genes: ancient intron in ancient gene, later-evolved intron in ancient gene, and later-evolved intron in later-evolved gene, which can reflect to some extent the dynamic evolution of introns in Giardia. Without finding any special features or functional importance of these introns responsible for their retention, we noticed and experimentally verified that some intron-containing genes form sense-antisense gene pairs with transcribable genes on their complementary strands, and that the introns just reside in the overlapping regions.ConclusionsIn Giardia’s evolution, despite constant evolutionary selection pressure of intron loss, intron gain can still occur in both ancient and later-evolved genes, but only a few introns are retained; at least the evolutionary retention of some of the introns might not be due to the functional constraint of the introns themselves but the causes outside of introns, such as the constraints imposed by other genomic functional elements overlapping with the introns. These findings can not only provide some clues to find new genomic functional elements -- in the areas overlapping with introns, but suggest that “functional constraint” of introns may not be necessarily directly associated with intron loss and gain, and that the real functions are probably still outside of our current knowledge.ReviewersThis article was reviewed by Mikhail Gelfand, Michael Gray, and Igor Rogozin.

Bioinformatic analysis of regulation of natural antisense transcripts by transposable elements in human mRNA

Non-coding RNA is no longer considered to be “junk” DNA, based on evidence uncovered in recent decades. In particular, the important role played by natural antisense transcripts (NATs) in regulating the expression of genes is receiving increasing attention. However, the regulatory mechanisms of NATs remain incompletely understood. It is well-known that the insertion of transposable elements (TEs) can affect gene transcription. Using a bioinformatics approach, we identified NATs using human mRNA sequences from the UCSC Genome Browser Database. Our in silico analysis identified 1079 NATs and 700 sense-antisense gene pairs. We identified 179 NATs that showed evidence of having been affected by TEs during cellular gene expression. These findings may provide an understanding of the complex regulation mechanisms of NATs. If our understanding of NATs as modulators of gene expression is further enhanced, we can develop ways to control gene expression.

Antisense Long Non-Coding RNAs Are Deregulated in Skin Tissue of Patients with Systemic Sclerosis

Systemic sclerosis is an autoimmune disease characterized by fibrosis of skin and multiple organs of which the pathogenesis is poorly understood. We studied differentially expressed coding and non-coding genes in relation to systemic sclerosis pathogenesis with a specific focus on antisense non-coding RNAs. Skin biopsy-derived RNAs from 14 early systemic sclerosis patients and six healthy individuals were sequenced with ion-torrent and analyzed using DEseq2. Overall, 4,901 genes with a fold change >1.5 and a false discovery rate <5% were detected in patients versus controls. Upregulated genes clustered in immunologic, cell adhesion, and keratin-related processes. Interestingly, 676 deregulated non-coding genes were detected, 257 of which were classified as antisense genes. Sense genes expressed opposite of these antisense genes were also deregulated in 42% of the observed sense-antisense gene pairs. The majority of the antisense genes had a similar effect sizes in an independent North American dataset with three genes (CTBP1-AS2, OTUD6B-AS1, and AGAP2-AS1) exceeding the study-wide Bonferroni-corrected P-value (PBonf < 0.0023, Pcombined= 1.1× 10-9, 1.4× 10-8, 1.7× 10-6, respectively). In this study, we highlight that together with coding genes, (antisense) long non-coding RNAs are deregulated in skin tissue of systemic sclerosis patients suggesting a novel class of genes involved in pathogenesis of systemic sclerosis.

A cautionary tale of sense-antisense gene pairs: independent regulation despite inverse correlation of expression.

Long non-coding RNAs (lncRNAs) have been proven to play important roles in diverse cellular processes including the DNA damage response. Nearly 40% of annotated lncRNAs are transcribed in antisense direction to other genes and have often been implicated in their regulation via transcript- or transcription-dependent mechanisms. However, it remains unclear whether inverse correlation of gene expression would generally point toward a regulatory interaction between the genes. Here, we profiled lncRNA and mRNA expression in lung and liver cancer cells after exposure to DNA damage. Our analysis revealed two pairs of mRNA-lncRNA sense-antisense transcripts being inversely expressed upon DNA damage. The lncRNA NOP14-AS1 was strongly upregulated upon DNA damage, while the mRNA for NOP14 was downregulated, both in a p53-dependent manner. For another pair, the lncRNA LIPE-AS1 was downregulated, while its antisense mRNA CEACAM1 was upregulated. To test whether as expected the antisense genes would regulate each other resulting in this highly significant inverse correlation, we employed antisense oligonucleotides and RNAi to study transcript-dependent effects as well as dCas9-based transcriptional modulation by CRISPRi/CRISPRa for transcription-dependent effects. Surprisingly, despite the strong stimulus-dependent inverse correlation, our data indicate that neither transcript- nor transcription-dependent mechanisms explain the inverse regulation of NOP14-AS1:NOP14 or LIPE-AS1:CEACAM1 expression. Hence, sense-antisense pairs whose expression is strongly—positively or negatively—correlated can be nonetheless regulated independently. This highlights the requirement of individual experimental studies for each antisense pair and prohibits drawing conclusions on regulatory mechanisms from expression correlations.

Sense-antisense gene-pairs in breast cancer and associated pathological pathways.

More than 30% of human protein-coding genes form hereditary complex genome architectures composed of sense-antisense (SA) gene pairs (SAGPs) transcribing their RNAs from both strands of a given locus. Such architectures represent important novel components of genome complexity contributing to gene expression deregulation in cancer cells. Therefore, the architectures might be involved in cancer pathways and, in turn, be used for novel drug targets discovery. However, the global roles of SAGPs in cancer pathways has not been studied. Here we investigated SAGPs associated with breast cancer (BC)-related pathways using systems biology, prognostic survival and experimental methods. Gene expression analysis identified 73 BC-relevant SAGPs that are highly correlated in BC. Survival modelling and metadata analysis of the 1161 BC patients allowed us to develop a novel patient prognostic grouping method selecting the 12 survival-significant SAGPs. The qRT-PCR-validated 12-SAGP prognostic signature reproducibly stratified BC patients into low- and high-risk prognostic subgroups. The 1381 SAGP-defined differentially expressed genes common across three studied cohorts were identified. The functional enrichment analysis of these genes revealed the GABPA gene network, including BC-relevant SAGPs, specific gene sets involved in cell cycle, spliceosomal and proteasomal pathways. The co-regulatory function of GABPA in BC cells was supported using siRNA knockdown studies. Thus, we demonstrated SAGPs as the synergistically functional genome architectures interconnected with cancer-related pathways and associated with BC patient clinical outcomes. Taken together, SAGPs represent an important component of genome complexity which can be used to identify novel aspects of coordinated pathological gene networks in cancers.

Chromatin-Mediated Reversible Silencing of Sense-Antisense Gene Pairs in Embryonic Stem Cells Is Consolidated upon Differentiation.

Genome-wide gene expression studies have indicated that the eukaryotic genome contains many gene pairs showing overlapping sense and antisense transcription. Regulation of these coding and/or noncoding gene pairs involves intricate regulatory mechanisms. In the present study, we utilized an enhanced green fluorescent protein (EGFP)-tagged reporter plasmid cis linked to a doxycycline-inducible antisense promoter, generating antisense transcription that fully overlaps EGFP, to study the mechanism and dynamics of gene silencing after induction of noncoding antisense transcription in undifferentiated and differentiating mouse embryonic stem cells (ESCs). We found that EGFP silencing is reversible in ESCs but is locked into a stable state upon ESC differentiation. Reversible silencing in ESCs is chromatin dependent and is associated with accumulation of trimethylated lysine 36 on histone H3 (H3K36me3) at the EGFP promoter region. In differentiating ESCs, antisense transcription-induced accumulation of H3K36me3 was associated with an increase in CpG methylation at the EGFP promoter. Repression of the sense promoter was affected by small-molecule inhibitors which interfere with DNA methylation and histone demethylation pathways. Our results indicate a general mechanism for silencing of fully overlapping sense-antisense gene pairs involving antisense transcription-induced accumulation of H3K36me3 at the sense promoter, resulting in reversible silencing of the sense partner, which is stabilized during ESC differentiation by CpG methylation.

Sense-antisense gene pairs: sequence, transcription, and structure are not conserved between human and mouse

Previous efforts to characterize conservation between the human and mouse genomes focused largely on sequence comparisons. These studies are inherently limited because they don't account for gene structure differences, which may exist despite genomic sequence conservation. Recent high-throughput transcriptome studies have revealed widespread and extensive overlaps between genes, and transcripts, encoded on both strands of the genomic sequence. This overlapping gene organization, which produces sense-antisense (SAS) gene pairs, is capable of effecting regulatory cascades through established mechanisms. We present an evolutionary conservation assessment of SAS pairs, on three levels: genomic, transcriptomic, and structural. From a genome-wide dataset of human SAS pairs, we first identified orthologous loci in the mouse genome, then assessed their transcription in the mouse, and finally compared the genomic structures of SAS pairs expressed in both species. We found that approximately half of human SAS loci have single orthologous locations in the mouse genome; however, only half of those orthologous locations have SAS transcriptional activity in the mouse. This suggests that high human-mouse gene conservation overlooks widespread distinctions in SAS pair incidence and expression. We compared gene structures at orthologous SAS loci, finding frequent differences in gene structure between human and orthologous mouse SAS pair members. Our categorization of human SAS pairs with respect to mouse conservation of expression as well as structure points to limitations of mouse models. Gene structure differences, including at SAS loci, may account for some of the phenotypic distinctions between primates and rodents. Genes in non-conserved SAS pairs may contribute to evolutionary lineage-specific regulatory outcomes.

Comprehensive expressional analyses of antisense transcripts in colon cancer tissues using artificial antisense probes

BackgroundRecent studies have identified thousands of sense-antisense gene pairs across different genomes by computational mapping of cDNA sequences. These studies have shown that approximately 25% of all transcriptional units in the human and mouse genomes are involved in cis-sense-antisense pairs. However, the number of known sense-antisense pairs remains limited because currently available cDNA sequences represent only a fraction of the total number of transcripts comprising the transcriptome of each cell type.MethodsTo discover novel antisense transcripts encoded in the antisense strand of important genes, such as cancer-related genes, we conducted expression analyses of antisense transcripts using our custom microarray platform along with 2376 probes designed specifically to detect the potential antisense transcripts of 501 well-known genes suitable for cancer research.ResultsUsing colon cancer tissue and normal tissue surrounding the cancer tissue obtained from 6 patients, we found that antisense transcripts without poly(A) tails are expressed from approximately 80% of these well-known genes. This observation is consistent with our previous finding that many antisense transcripts expressed in a cell are poly(A)-. We also identified 101 and 71 antisense probes displaying a high level of expression specifically in normal and cancer tissues respectively.ConclusionOur microarray analysis identified novel antisense transcripts with expression profiles specific to cancer tissue, some of which might play a role in the regulatory networks underlying oncogenesis and thus are potential targets for further experimental validation. Our microarray data are available at http://www.brc.riken.go.jp/ncrna2007/viewer-Saito-01/index.html.