Antisense Transcripts Research Articles

Interplay between coding and non-coding regulation drives the Arabidopsis seed-to-seedling transition

Translation of seed stored mRNAs is essential to trigger germination. However, when RNAPII re-engages RNA synthesis during the seed-to-seedling transition has remained in question. Combining csRNA-seq, ATAC-seq and smFISH in Arabidopsis thaliana we demonstrate that active transcription initiation is detectable during the entire germination process. Features of non-coding regulation such as dynamic changes in chromatin accessible regions, antisense transcription, as well as bidirectional non-coding promoters are widespread throughout the Arabidopsis genome. We show that sensitivity to exogenous ABSCISIC ACID (ABA) during germination depends on proximal promoter accessibility at ABA-responsive genes. Moreover, we provide genetic validation of the existence of divergent transcription in plants. Our results reveal that active enhancer elements are transcribed producing non-coding enhancer RNAs (eRNAs) as widely documented in metazoans. In sum, this study defining the extent and role of coding and non-coding transcription during key stages of germination expands our understanding of transcriptional mechanisms underlying plant developmental transitions.

Economy of Effort or Sophisticated Programming? The Prevalence of Bidirectional Promoter Complexes in the Human Genome.

An abundance of antisense promoters in the vicinity of the transcriptional start site of coding genes suggests that they play an important role in gene regulation. The divergent transcription of housekeeping genes by a common central promoter region allows for coordinated regulation of genes in related pathways and is also linked to higher promoter activity. However, closely positioned transcription start sites can also result in competition between overlapping promoter elements and generate a binary switch element. Furthermore, the direct competition resulting from the presence of an antisense promoter immediately downstream of the transcription start site of the gene produces an element that can exist in only one of two stable transcriptional states: sense or antisense. In this review, we summarize analyses of the prevalence of antisense transcription in higher eukaryotes and viruses, with a focus on the antisense promoters competing with the promoters of coding genes. The structures of bidirectional promoters driving the simultaneous expression of housekeeping genes are compared with examples of human bidirectional elements that have been shown to act as switches. Since many bidirectional elements contain a noncoding RNA as the divergent transcript, we describe examples of functional noncoding antisense transcripts that affect the epigenetic landscape and alter the expression of their host gene. Finally, we discuss opportunities for additional research on competing sense/antisense promoters, uncovering their potential role in programming cell differentiation.

Transcriptome analysis of Edwardsiella piscicida during intracellular infection reveals excludons are involved with the activation of a mitochondrion-like energy generation program.

Phylogenetic evidence suggests a shared ancestry between mitochondria and modern Proteobacteria, a phylum including several genera of intracellular pathogens. Studying these diverse pathogens, particularly during intracellular infection of their hosts, can reveal characteristics potentially representative of the mitochondrial-Proteobacterial ancestor by identifying traits shared with mitochondria. While transcriptomic approaches can provide global insights into intracellular acclimatization by pathogens, they are often limited by excess host RNAs in extracts. Here, we developed a method employing magnetic nanoparticles to enrich RNA from an intracellular Gammaproteobacterium, Edwardsiella piscicida, within zebrafish, Danio rerio, fin fibroblasts, enabling comprehensive exploration of the bacterial transcriptome. Our findings revealed that the intracellular E. piscicida transcriptome reflects a mitochondrion-like energy generation program characterized by the suppression of glycolysis and sugar transport, coupled with upregulation of the tricarboxylic acid (TCA) cycle and alternative import of simple organic acids that directly flux into TCA cycle intermediates or electron transport chain donors. Additionally, genes predicted to be members of excludons, loci of gene pairs antagonistically co-regulated by overlapping antisense transcription, are significantly enriched in the set of all genes with perturbed sense and antisense transcription, suggesting a general but important involvement of excludons with intracellular acclimatization. Notably, genes involved with the activation of the mitochondrion-like energy generation program, specifically with metabolite import and glycolysis, are also members of predicted excludons. Other intracellular Proteobacterial pathogens appear to employ a similar mitochondrion-like energy generation program, suggesting a potentially conserved mechanism for optimized energy acquisition from hosts centered around the TCA cycle.IMPORTANCEPhylogenetic evidence suggests that mitochondria and Proteobacteria, a phylum encompassing various intracellular pathogens, share a common ancestral lineage. In this study, we developed a novel method employing magnetic nanoparticles to explore the transcriptome of an aquatic Gammaproteobacterium, Edwardsiella piscicida, during intracellular infection of host cells. We show that the strategy E. piscicida uses to generate energy strikingly mirrors the function of mitochondria-energy generators devoid of glycolytic processes. Notably, several implicated genes are members of excludons-gene pairs antagonistically co-regulated by overlapping antisense transcription. Other intracellular Proteobacterial pathogens appear to adopt a similar mitochondrion-like energy generation program, indicating a possibly conserved strategy for optimized energy acquisition from hosts centered around the tricarboxylic acid cycle.

Regulation of TRI5 expression and deoxynivalenol biosynthesis by a long non-coding RNA in Fusarium graminearum

Deoxynivalenol (DON) is the most frequently detected mycotoxin in cereal grains and processed food or feed. Two transcription factors, Tri6 and Tri10, are essential for DON biosynthesis in Fusarium graminearum. In this study we conduct stranded RNA-seq analysis with tri6 and tri10 mutants and show that Tri10 acts as a master regulator controlling the expression of sense and antisense transcripts of TRI6 and over 450 genes with diverse functions. TRI6 is more specific for regulating TRI genes although it negatively regulates TRI10. Two other TRI genes, including TRI5 that encodes a key enzyme for DON biosynthesis, also have antisense transcripts. Both Tri6 and Tri10 are essential for TRI5 expression and for suppression of antisense-TRI5. Furthermore, we identify a long non-coding RNA (named RNA5P) that is transcribed from the TRI5 promoter region and is also regulated by Tri6 and Tri10. Deletion of RNA5P by replacing the promoter region of TRI5 with that of TRI12 increases TRI5 expression and DON biosynthesis, indicating that RNA5P suppresses TRI5 expression. However, ectopic constitutive overexpression of RNA5P has no effect on DON biosynthesis and TRI5 expression. Nevertheless, elevated expression of RNA5P in situ reduces TRI5 expression and DON production. Our results indicate that TRI10 and TRI6 regulate each other’s expression, and both are important for suppressing the expression of RNA5P, a long non-coding RNA with cis-acting inhibitory effects on TRI5 expression and DON biosynthesis in F. graminearum.

Prenatal delivery of a therapeutic antisense oligonucleotide achieves broad biodistribution in the brain and ameliorates Angelman syndrome phenotype in mice

Angelman syndrome (AS), an early-onset neurodevelopmental disorder characterized by abnormal gait, intellectual disabilities, and seizures, occurs when the maternal allele of the UBE3A gene is disrupted, since the paternal allele is silenced in neurons by the UBE3A antisense (UBE3A-AS) transcript. Given the importance of early treatment, we hypothesized that prenatal delivery of an antisense oligonucleotide (ASO) would downregulate the murine Ube3a-AS, resulting in increased UBE3A protein and functional rescue. Using a mouse model with a Ube3a-YFP allele that reports on-target ASO activity, we found that in utero, intracranial (IC) injection of the ASO resulted in dose-dependent activation of paternal Ube3a, with broad biodistribution. Accordingly, in utero injection of the ASO in a mouse model of AS also resulted in successful restoration of UBE3A and phenotypic improvements in treated mice on the accelerating rotarod and fear conditioning. Strikingly, even intra-amniotic (IA) injection resulted in systemic biodistribution and high levels of UBE3A reactivation throughout the brain. These findings offer a novel strategy for early treatment of AS using an ASO, with two potential routes of administration in the prenatal window. Beyond AS, successful delivery of a therapeutic ASO into neurons has implications for a clinically feasible prenatal treatment for numerous neurodevelopmental disorders.

Tyrosine 1-phosphorylated RNA polymerase II transcribes PROMPTs to facilitate proximal promoter pausing and induce global transcriptional repression in response to DNA damage.

DNA damage triggers a complex transcriptional response that involves both activation and repression of gene expression. In this study, we investigated global changes in transcription in response to ionizing irradiation (IR), which induces double-strand breaks in DNA. We used mNET-seq to profile nascent transcripts bound to different phosphorylated forms of the RNA polymerase II (RNA Pol II) C-terminal domain (CTD). We found that IR leads to global transcriptional repression of protein-coding genes, accompanied by an increase in antisense transcripts near promoters, called PROMPTs, transcribed by RNA Pol II phosphorylated on tyrosine 1 (Y1P) residue of the CTD. These Y1P-transcribed PROMPTs are enriched for PRC2 binding sites and associated with RNA Pol II proximal promoter pausing. We show the interaction between Y1P RNA Pol II and PRC2, as well as PRC2 binding to PROMPTs. Inhibition of PROMPTs or depletion of PRC2 leads to loss of transcriptional repression. Our results reveal a novel function of Y1P-dependent PROMPTs in mediating PRC2 recruitment to chromatin and RNA Pol II promoter pausing in response to DNA damage.

A Revision of Herpes Simplex Virus Type 1 Transcription: First, Repress; Then, Express.

The herpes virus genome bears more than 80 strong transcriptional promoters. Upon entry into the host cell nucleus, these genes are transcribed in an orderly manner, producing five immediate-early (IE) gene products, including ICP0, ICP4, and ICP22, while non-IE genes are mostly silent. The IE gene products are necessary for the transcription of temporal classes following sequentially as early, leaky late, and true late. A recent analysis using precision nuclear run-on followed by deep sequencing (PRO-seq) has revealed an important step preceding all HSV-1 transcription. Specifically, the immediate-early proteins ICP4 and ICP0 enter the cell with the incoming genome to help preclude the nascent antisense, intergenic, and sense transcription of all viral genes. VP16, which is also delivered into the nucleus upon entry, almost immediately reverses this repression on IE genes. The resulting de novo expression of ICP4 and ICP22 further repress antisense, intergenic, and early and late viral gene transcription through different mechanisms before the sequential de-repression of these gene classes later in infection. This early repression, termed transient immediate-early protein-mediated repression (TIEMR), precludes unproductive, antisense, intergenic, and late gene transcription early in infection to ensure the efficient and orderly progression of the viral cascade.

COOLAIR and PRC2 function in parallel to silence FLC during vernalization

Noncoding transcription induces chromatin changes that can mediate environmental responsiveness, but the causes and consequences of these mechanisms are still unclear. Here, we investigate how antisense transcription (termed COOLAIR) interfaces with Polycomb Repressive Complex 2 (PRC2) silencing during winter-induced epigenetic regulation of Arabidopsis FLOWERING LOCUS C (FLC). We use genetic and chromatin analyses on lines ineffective or hyperactive for the antisense pathway in combination with computational modeling to define the mechanisms underlying FLC repression. Our results show that FLC is silenced through pathways that function with different dynamics: a COOLAIR transcription-mediated pathway capable of fast response and in parallel a slow PRC2 switching mechanism that maintains each allele in an epigenetically silenced state. Components of both the COOLAIR and PRC2 pathways are regulated by a common transcriptional regulator (NTL8), which accumulates by reduced dilution due to slow growth at low temperature. The parallel activities of the regulatory steps, and their control by temperature-dependent growth dynamics, create a flexible system for registering widely fluctuating natural temperature conditions that change year on year, and yet ensure robust epigenetic silencing of FLC.

Recent insights into the functions and mechanisms of antisense RNA: emerging applications in cancer therapy and precision medicine.

The antisense RNA molecule is a unique DNA transcript consisting of 19-23 nucleotides, characterized by its complementary nature to mRNA. These antisense RNAs play a crucial role in regulating gene expression at various stages, including replication, transcription, and translation. Additionally, artificial antisense RNAs have demonstrated their ability to effectively modulate gene expression in host cells. Consequently, there has been a substantial increase in research dedicated to investigating the roles of antisense RNAs. These molecules have been found to be influential in various cellular processes, such as X-chromosome inactivation and imprinted silencing in healthy cells. However, it is important to recognize that in cancer cells; aberrantly expressed antisense RNAs can trigger the epigenetic silencing of tumor suppressor genes. Moreover, the presence of deletion-induced aberrant antisense RNAs can lead to the development of diseases through epigenetic silencing. One area of drug development worth mentioning is antisense oligonucleotides (ASOs), and a prime example of an oncogenic trans-acting long noncoding RNA (lncRNA) is HOTAIR (HOX transcript antisense RNA). NATs (noncoding antisense transcripts) are dysregulated in many cancers, and researchers are just beginning to unravel their roles as crucial regulators of cancer's hallmarks, as well as their potential for cancer therapy. In this review, we summarize the emerging roles and mechanisms of antisense RNA and explore their application in cancer therapy.

Expression profiles of the lncRNA antisense GAS5-AS1 in colon biopsies from pediatric inflammatory bowel disease patients and its role in regulating sense transcript GAS5.

The long non-coding RNA (lncRNA) growth arrest-specific transcript 5 (GAS5) level was demonstrated as involved in pediatric inflammatory bowel disease (IBD) pathogenesis. Since its antisense transcript GAS5-AS1 has never been investigated in IBD, this study aims to detect whether GAS5-AS1 and GAS5 levels are related to IBD clinical parameters and investigate their correlation in vitro. Twenty-six IBD pediatric patients were enrolled; paired inflamed and non-inflamed intestinal biopsies were collected. We evaluated GAS5 and GAS5-AS1 levels by real-time PCR. The role of GAS5 and GAS5-AS1 was assessed in vitro by transient silencing in THP1-derived macrophages. GAS5-AS1 and GAS5 levels were associated with patients' clinical parameters; GAS5-AS1 expression was downregulated in inflamed tissues and inversely correlated with disease activity. A positive correlation between GAS5-AS1 and GAS5 levels was observed in non-inflamed biopsies. On THP1-derived macrophages, a reduced amount of both GAS5-AS1 and GAS5 was observed; accordingly, matrix metalloproteinase (MMP) 9 was increased. After GAS5-AS1 silencing, a downregulation of GAS5 was found, whereas no effect was detected on GAS5-AS1 after GAS5 silencing. Conclusion: This study provided for the first time new insights into the potential role of GAS5-AS1 in IBD. GAS5-AS1 modulates GAS5 levels in vitro and may serve as a potential IBD diagnostic biomarker. What is Known: • GAS5 is involved in regulating intestinal MMP-2 and MMP-9 in pediatric patients with IBD; • GAS5-AS1 has never been investigated in the context of IBD; • GAS5-AS1 regulates the expression of GAS5, increasing its stability in tissues and in vitro cell models of cancer. What is New: • GAS5-AS1 correlated with GAS5 and IBD clinical parameters; • GAS5-AS1 can modulate GAS5 levels in macrophages; • GAS5-AS1 may serve as potential IBD diagnostic biomarker.

Current perspectives of lncRNAs in abiotic and biotic stress tolerance in plants.

Abiotic/biotic stresses pose a major threat to agriculture and food security by impacting plant growth, productivity and quality. The discovery of extensive transcription of large RNA transcripts that do not code for proteins, termed long non-coding RNAs (lncRNAs) with sizes larger than 200 nucleotides in length, provides an important new perspective on the centrality of RNA in gene regulation. In plants, lncRNAs are widespread and fulfill multiple biological functions in stress response. In this paper, the research advances on the biological function of lncRNA in plant stress response were summarized, like as Natural Antisense Transcripts (NATs), Competing Endogenous RNAs (ceRNAs) and Chromatin Modification etc. And in plants, lncRNAs act as a key regulatory hub of several phytohormone pathways, integrating abscisic acid (ABA), jasmonate (JA), salicylic acid (SA) and redox signaling in response to many abiotic/biotic stresses. Moreover, conserved sequence motifs and structural motifs enriched within stress-responsive lncRNAs may also be responsible for the stress-responsive functions of lncRNAs, it will provide a new focus and strategy for lncRNA research. Taken together, we highlight the unique role of lncRNAs in integrating plant response to adverse environmental conditions with different aspects of plant growth and development. We envisage that an improved understanding of the mechanisms by which lncRNAs regulate plant stress response may further promote the development of unconventional approaches for breeding stress-resistant crops.

Regional and cellular organization of the autism-associated protein UBE3A/E6AP and its antisense transcript in the brain of the developing rhesus monkey.

Angelman syndrome (AS) is a neurogenetic disorder caused by mutations or deletions in the maternally-inherited UBE3A allele, leading to a loss of UBE3A protein expression in neurons. The paternally-inherited UBE3A allele is epigenetically silenced in neurons during development by a noncoding transcript (UBE3A-ATS). The absence of neuronal UBE3A results in severe neurological symptoms, including speech and language impairments, intellectual disability, and seizures. While no cure exists, therapies aiming to restore UBE3A function-either by gene addition or by targeting UBE3A-ATS-are under development. Progress in developing these treatments relies heavily on inferences drawn from mouse studies about the function of UBE3A in the human brain. To aid translational efforts and to gain an understanding of UBE3A and UBE3A-ATS biology with greater relevance to human neurodevelopmental contexts, we investigated UBE3A and UBE3A-ATS expression in the developing brain of the rhesus macaque, a species that exhibits complex social behaviors, resembling aspects of human behavior to a greater degree than mice. Combining immunohistochemistry and in situ hybridization, we mapped UBE3A and UBE3A-ATS regional and cellular expression in normal prenatal, neonatal, and adolescent rhesus macaque brains. We show that key hallmarks of UBE3A biology, well-known in rodents, are also present in macaques, and suggest paternal UBE3A silencing in neurons-but not glial cells-in the macaque brain, with onset between gestational day 48 and 100. These findings support proposals that early-life, perhaps even prenatal, intervention is optimal for overcoming the maternal allele loss of UBE3A linked to AS.

PAPAS promotes differentiation of mammary epithelial cells and suppresses breast carcinogenesis

Extensive remodeling of the female mammary epithelium during development and pregnancy has been linked to cancer susceptibility. The faithful response of mammary epithelial cells (MECs) to hormone signaling is key to avoiding breast cancer development. Here, we show that lactogenic differentiation of murine MECs requires silencing of genes encoding ribosomal RNA (rRNA) by the antisense transcript PAPAS. Accordingly, knockdown of PAPAS derepresses rRNA genes, attenuates the response to lactogenic hormones, and induces malignant transformation. Restoring PAPAS levels in breast cancer cells reduces tumorigenicity and lung invasion and activates many interferon-regulated genes previously linked to metastasis suppression. Mechanistically, PAPAS transcription depends on R-loop formation at the 3' end of rRNA genes, which is repressed by RNase H1 and replication protein A (RPA) overexpression in breast cancer cells. Depletion of PAPAS and upregulation of RNase H1 and RPA in human breast cancer underpin the clinical relevance of our findings.

De novo transcriptome assembly of mouse male germ cells reveals novel genes, stage-specific bidirectional promoter activity, and noncoding RNA expression.

In mammals, the adult testis is the tissue with the highest diversity in gene expression. Much of that diversity is attributed to germ cells, primarily meiotic spermatocytes and postmeiotic haploid spermatids. Exploiting a newly developed cell purification method, we profiled the transcriptomes of such postmitotic germ cells of mice. We used a de novo transcriptome assembly approach and identified thousands of novel expressed transcripts characterized by features distinct from those of known genes. Novel loci tend to be short in length, monoexonic, and lowly expressed. Most novel genes have arisen recently in evolutionary time and possess low coding potential. Nonetheless, we identify several novel protein-coding genes harboring open reading frames that encode proteins containing matches to conserved protein domains. Analysis of mass-spectrometry data from adult mouse testes confirms protein production from several of these novel genes. We also examine overlap between transcripts and repetitive elements. We find that although distinct families of repeats are expressed with differing temporal dynamics during spermatogenesis, we do not observe a general mode of regulation wherein repeats drive expression of nonrepetitive sequences in a cell type-specific manner. Finally, we observe many fairly long antisense transcripts originating from canonical gene promoters, pointing to pervasive bidirectional promoter activity during spermatogenesis that is distinct and more frequent compared with somatic cells.

Selective RNA‐targeting of mTORC1 and 2 to ameliorate Alzheimer’s Disease pathogenesis

AbstractBackgroundIt has been well described that inhibition of the kinase, mechanistic target of rapamycin (mTOR), improves age‐related pathologies including Alzheimer’s Disease (AD). However, mTOR exists as a complex and exerts differential functions depending on its binding partners. Suppression of mTORC1 has yielded positive outcomes for AD‐related measures (decreased amyloid beta/tau pathologies and improved cognition)1. In contrast, increased mTORC2 activity also resulted in positive outcomes in AD models (improved brain insulin sensitivity and neuronal survival)2, suggesting that a therapeutic which inhibits mTORC1 while upregulating mTORC2 may be desirable for AD. However, no such therapeutic exists.MethodTo inhibit mTORC1, we used a raptor siRNA which has been chemically modified to be stable for in vivo purposes. To upregulate mTORC2, we bioinformatically identified natural antisense transcripts (NATs) of rictor and designed custom siRNAs to target the various transcripts. Following an in vitro screen of the custom siRNAs, we determined which NATs yield upregulation of rictor when targeted and designed more siRNAs targeting the same transcript to optimize our mTORC2‐upregulating siRNA. Cellular RNA and protein were quantified using RT‐qPCR and western blot, respectively.ResultWe demonstrate efficient knockdown of raptor in all transfected cell lines, as well as in mouse brain. Raptor knockdown has been confirmed at both the RNA and protein level. Furthermore, we successfully upregulated expression of rictor using rictor NAT‐targeting siRNAs (AntagoNATs) in multiple cell lines. Downstream analyses suggest a therapeutic effect for both raptor knockdown and rictor upregulation in the context of AD.ConclusionNucleic acid‐based therapeutics which target RNA have been outcompeting and replacing small molecule protein‐targeting drugs in recent years. However, RNA‐targeted therapeutics have been largely unexplored for AD. Chemical linkage of raptor siRNA to rictor AntagoNAT siRNA yields the first therapeutic to selectively inhibit mTORC1 and upregulate mTORC2, providing a modern RNA‐targeting therapeutic for AD.

Pathogenicity‐related long non‐coding natural antisense transcripts in Verticillium dahliae during infections in cotton

AbstractLong non‐coding RNAs (lncRNAs) are non‐coding RNAs with a length of more than 200 nucleotides. LncRNAs are functional factors involved in various biological processes at the transcriptional, post‐transcriptional and epigenetic regulation, which have been widely studied in animals and plants, but less in fungi. In our previous research, 352 Verticillium dahliae lncRNAs were identified by high‐throughput sequencing, and pathogenicity‐related lncRNAs were determined by their differential expression during infections on host plants of cotton. It is known that a long non‐coding natural antisense transcript (lncNAT) transcribed from the antisense strand of a gene, mainly plays cis‐regulatory functions. Thus, in this study, V. dahliae lncNATs and their function were further analysed based on our previous research of V. dahliae lncRNAs. In total, 116 of 352 lncRNAs were identified as lncNATs, and the function of their associated genes that are mainly enriched in transcriptional regulation was assessed. Moreover, 12 lncNATs were significantly differentially expressed during infection in cotton, and their associated genes are involved in its pathogenicity. In addition, the expression pattern between lncNAT010781 and lncNAT005061 and their associated genes were detected. Both lncNATs and their associated genes were up‐regulated during the inoculation of cotton. Overexpression of lncNAT005061 enhanced the virulence of Vd991 on cotton. In conclusion, these results suggest that V. dahliae lncNATs play a pathogenic role when cotton host plants are infected, which will provide new targets for analysing the virulence regulatory network in V. dahliae.

Molecular cloning, expression and cellular localization of two long noncoding RNAs (mLINC-RBE and mLINC-RSAS) in the mouse testis

Long noncoding RNAs (lncRNAs) are transcribed in complex, overlapping, sense- and antisense orientations from intronic and intergenic regions of mammalian genomes. Transcription of genome in mammalian testis is more widespread compared to other organs. LncRNAs are involved in gene expression, chromatin regulation, mRNA stability and translation of proteins during diverse cellular functions. We report molecular cloning of two novel lncRNAs (mLINC-RBE and mLINC-RSAS) and their expression by RT-PCR as well as cellular localization by RNA in-situ hybridization in the mouse testes. mLINC-RBE is an intergenic lncRNA from chromosome 4, with 16.96 % repeat sequences, expressed as a sense transcript with piRNA sequences and its expression is localized into primary spermatocytes. mLINC-RSAS is an intergenic lncRNA from chromosome 2, with 49.7 % repeat sequences, expressed as both sense- and antisense transcripts with miRNA sequences and its expression is localized into different cell types, such as Sertoli cells, primary spermatocytes and round spermatids. The lncRNAs also contain sequences for some short peptides (micropeptides). This suggests that these two repeat sequence containing, intergenic genomic sense- and antisense transcripts expressed as lncRNAs with piRNAs, miRNAs, and showing cell-type specific, differential expression may regulate important functions in mammalian testes. Such functions may be regulated by RNA structures, RNA processing and RNA-protein complexes.

LADON, a Natural Antisense Transcript of NODAL, Promotes Tumour Progression and Metastasis in Melanoma.

The TGFβ family member NODAL, repeatedly required during embryonic development, has also been associated with tumour progression. Our aim was to clarify the controversy surrounding its involvement in melanoma tumour progression. We found that the deletion of the NODAL exon 2 in a metastatic melanoma cell line impairs its ability to form tumours and colonize distant tissues. However, we show that this phenotype does not result from the absence of NODAL, but from a defect in the expression of a natural antisense transcript of NODAL, here called LADON. We show that LADON expression is specifically activated in metastatic melanoma cell lines, that its transcript is packaged in exosomes secreted by melanoma cells, and that, via its differential impact on the expression of oncogenes and tumour suppressors, it promotes the mesenchymal to amoeboid transition that is critical for melanoma cell invasiveness. LADON is, therefore, a new player in the regulatory network governing tumour progression in melanoma and possibly in other types of cancer.

Endothelial cell-expressed long non-coding RNAs in cardiac regeneration

Abstract Endothelial cells (ECs) are the most abundant non-myocyte cell types in the heart. Upon cardiac injury, revascularization can occur through proliferation and migration of ECs from existing blood vessels in the surrounding myocardium. In animal models, revascularization is crucial to stimulate cardiomyocyte survival and regeneration. In the human heart, establishing blood flow could improve outcomes of tissue engineering or cell based therapy to regenerate the heart after injury. In addition to establishing blood flow, EC also secrete factors in a paracrine manner which can act on neighbouring cardiomyocytes. We investigate a role for non-coding RNA in the regulation of endothelial cell function and their potential role in neovascularization and cardiac regeneration. A large portion of the genome is transcribed into non-coding RNA, which does not encode protein. Many long non-coding RNAs (lncRNAs) have been shown to be involved in important regulatory processes such as genomic imprinting and chromatin modification. To identify lncRNAs which could be involved in cardiac regeneration, we isolated ECs from neonatal mice at postnatal day 1 and 5.5. The potential for cardiac regeneration was observed in mice at postnatal day 1, but this capacity is lost by day 5.5. Differential expression of lncRNAs in ECs was assessed by RNA sequencing. Three potential target lncRNAs, which also have a human homologue, were selected. NR2F2-AS1 and HAND2-AS1 were upregulated at postnatal day 1, whereas FOXD2-AS1 was upregulated at day 5.5. These genes were selected for further validation in human cardiac microvascular endothelial cells (CMEC). Knockdown of NR2F2-AS1 resulted in a decrease in proliferation but did not affect angiogenic sprouting. Loss of HAND2-AS1 resulted in a trend to impaired sprouting but did not affect proliferation. FOXD2-AS1 knockdown resulted in a significant decrease in angiogenic sprouting. We observed no change in the rate of migration after knockdown of any of the three genes. Knockdown of the lncRNAs did not affect expression of the antisense protein coding transcripts, therefore the effects we observe are likely mediated by the lncRNAs. In conclusion, lncRNAs NR2F2-AS1, HAND2-AS1 and FOXD2-AS1 regulate different aspects of EC function such as proliferation and sprouting. Modulation of expression of these transcripts therefore may be considered as a therapeutic tool to promote cardiac regeneration.

GATA1 and GATA2 Sense/Antisense Transcriptional Switching in Early Hematopoietic Development

The GATA1 and GATA2 genes play essential roles in hematopoietic development. GATA1 is the master regulator of erythropoiesis and GATA2 is required for the expansion and survival of hematopoietic progenitor and stem cells, as well as the development of mast cells. A switch in GATA expression occurs during erythropoiesis, with silencing of the GATA2 gene and increased expression of GATA1. A GATA2 antisense transcript ( GATA2-AS1) has been associated with silencing of GATA2 expression. We have identified a novel GATA1 antisense promoter located in the first intron that opposes the GATA1 promoter and generates a spliced antisense transcript complementary to the SUV39H1 H3K9 methyltransferase gene. The competition between opposing sense and antisense promotersin the GATA1 and GATA2 genes may play a central role in the switching of GATA expression that occurs during hematopoiesis. To address this hypothesis, gene expression analysis of cells expressing sense versus antisense transcripts from the GATA1 or GATA2 genes was performed using 5'scRNAseq data from in vitro differentiated CD34 cells. Cells expressing only sense or only antisense GATA1 transcriptswere readily detected and comprised the majority (90%) of cells with GATA1 transcripts. Differential gene expression analysis of cells with GATA1 sense-only versus cells with GATA1 antisense-only showed association of sense transcription with erythrocyte genes, whereas antisense expression was associated with a mast cell phenotype and upregulation of GATA2 transcript levels. Conversely, GATA2 antisense expressing cells had increased GATA1 transcription and an erythrocyte phenotype, and GATA2 sense expressing cells demonstrated mast cell-specific gene expression. Single cell RNAseq analysis of HL60 cells induced to differentiate in vitro identified significant changes in GATA2 gene expression. Induction of granulocyte differentiation in HL60 cells treated with DMSO resulted in increased GATA2 antisense transcription, whereas PMA induction of monocyte differentiation was associated with increased GATA2 sense transcription. Taken together these results indicate a central role for sense/antisense promoter switching in hematopoiesis.