Endogenous Antisense Transcripts Research Articles

A novel long noncoding RNA lnc158 promotes the differentiation of mouse neural precursor cells into oligodendrocytes by targeting nuclear factor-IB.

Long noncoding RNAs have been implicated in oligodendrocyte myelination and oligodendrocyte maturation, but their roles in normal oligodendrocyte differentiation are not fully defined. Here, we report a novel nonprotein-coding RNA, named lnc158, discovered in mouse oligodendrocytes identified in subependymal ventricular zone tissue by single-cell RNA sequencing. Lnc158 is an endogenous antisense transcript of nuclear factor-IB (NFIB) and complementary to 3' untranslated region of NFIB mRNA. NFIB is a member of the nuclear factor-I family and is essential in the development of many organs such as brains and lungs. We found that lnc158 transcripts serve a biological function by regulating the transcription level of the NFIB coding gene in neural stem cells. Overexpression of lnc158 increased the expression of NFIB mRNA and knockdown of lnc158 decreased the expression of NFIB mRNA, suggesting that NFIB is regulated positively by lnc158. Further analyses showed that overexpression of lnc158 in neural stem cells induced a modest increase in CNP, MBP, MAG, and OSP mRNA level, and enhanced induction of differentiation along the lineage of oligodendrocytes. These results together imply that lnc158 positively modulates the transcription level of NFIB mRNA, leading to the enhanced induction of oligodendrocytes.

Small RNA-directed epigenetic programming of embryonic stem cell cardiac differentiation

Microinjection of small noncoding RNAs in one-cell embryos was reported in several instances to result in transcriptional activation of target genes. To determine the molecular mechanisms involved and to explore whether such epigenetic regulations could play a role in early development, we used a cell culture system as close as possible to the embryonic state. We report efficient cardiac differentiation of embryonic stem (ES) cells induced by small non-coding RNAs with sequences of Cdk9, a key player in cardiomyocyte differentiation. Transfer of oligoribonucleotides representing parts of the Cdk9 mRNA into ES and mouse embryo fibroblast cultures resulted in upregulation of transcription. Dependency on Argonaute proteins and endogenous antisense transcripts indicated that the inducer oligoribonucleotides were processed by the RNAi machinery. Upregulation of Cdk9 expression resulted in increased efficiency of cardiac differentiation suggesting a potential tool for stem cell-based regenerative medicine.

Noncoding RNAs: the fourth dimension of the human genome

amount of the transcriptome in biology is however, unclear. This part of the genome, the noncoding RNAs (ncRNAs) can be rightly be considered the fourth dimension of the genome. The ncRNAs comprise the long non-coding RNAs (lncRNAs), the long intergenic RNAs (lincRNAs), endogenous antisense RNAs, the microRNAs (miRs) and pseudogenes-derived lncRNAs [7]. The current version of the GENCODE database (v 22) estimates there to be 25,794 ncRNAs in humans [11,12]. The ncRNAs are crucial in gene regulation exerting their regulatory role at the chromatin, transcriptome and proteome level [13]. The regulation of the target genes by the ncRNAs occurs at the level of RNA and protein stabilization, protein binding and protein translocation or processing [9].Originally thought to be non-protein coding transcripts, at least some of the ncRNAs now seem to be capable of coding for Open Reading Frames [14,15]. Thus, eventually the terminology non-coding will have to evolve to accurately reflect the differences between the coding and the non-coding nature of the transcriptome. Increasing evidence in the ncRNA field of research is beginning to challenge our current understanding of the human genome. Pseudogene-derived lncRNAs are emerging as key regulators of gene expression and as a reservoir for miRs and endogenous antisense transcripts [16]. In addition, various Genome Wide Association Studies (GWAS) have identified a vast majority of disease-associated Single Nucleotide Polymorphisms (SNPs) which are present at the non-coding intronic or intergenic regions of the genes [17,18]. These regions were neglected in the past because of the focus on the protein coding regions. However, the attention is beginning to shift to this unexplored region of the human genome. Understanding the impact of these SNPs is going to allow us to develop a better understanding of diverse disease-associated phenotypes in the future. The ncRNAs including the miRs are of major interest for novel therapeutics as well as diagnostic biomarker development [4,19,20]. Numerous tools are becoming available to study the ncRNAs [21-26]. A microRNAs miR 222, is being developed as a therapeutic for hepatocellular carcinoma [27]. The Federal Drug Agency (FDA) approved its first lncRNA, the Prostate cancer antigen 3 (PCA3) as a urinary biomarker for prostate cancer diagnosis [28]. The miRs because of the stability in body fluids due to the secretome nature involving the exosomes, offer tremendous opportunities for biomarker potential for diagnosis and prognosis for cancer and other diseases [19,29-31]. The ncRNAs are the next frontier in molecular biology. The fruits of research in the ncRNA arena in the next decade are likely to challenge the current dogma in molecular biology. As we begin to understand more about the role of the entire transcriptome in gene regulation, our understanding of biology and function of a gene will achieve greater clarity. This is likely to lead into novel therapy and diagnosis for diverse diseases.

RNA-based inhibitors of endogenous antisense transcripts for locus-specific gene upregulation

RNA-based inhibitors of endogenous antisense transcripts for locus-specific gene upregulation

Genome-wide impact of endogenous antisense transcripts in eukaryotes

Recent transcriptomic studies revealed that extensive proportions of genomes are transcribed, despite the limited fraction of protein-coding gene loci in the whole genome. Most transcripts are considered to be 'cryptic' output of the genome because of the lack of functional evidence; however, recent progress in molecular analyses has revealed that some of these transcripts at least have functional significance. This review article examines evidence of the functional significance of endogenous cis-antisense transcripts, which are the transcriptional output from the opposite strand of annotated genes. These transcripts are one of the most common types of transcripts that do not correspond to any protein-coding loci. Historical molecular studies revealed the existence of antisense transcripts associated with dozens of gene loci, whereas more recent genome-wide studies have shown that many genes have an antisense counterpart thus stimulating investigations into the functional significance of endogenous antisense transcripts. Here, we summarize the recent progress in the genome-wide characterization of the antisense transcriptome, and discuss the biological mechanisms that underlie the regulatory machinery of eukaryotic gene expression with respect to the potential roles of endogenous cis-antisense transcripts.

ES cell differentiation system recapitulates the establishment of imprinted gene expression in a cell-type-specific manner

Genomic imprinting is a phenomenon whereby monoallelic gene expression occurs in a parent-of-origin-specific manner. A subset of imprinted genes acquires a tissue-specific imprinted status during the course of tissue development, and this process can be analyzed by means of an in vitro differentiation system utilizing embryonic stem (ES) cells. In neurons, the gene Ube3a is expressed from the maternal allele only, and a paternally expressed non-coding, antisense RNA has been implicated in the imprinting process in mice and humans. Here, to study the genomic imprinting mechanism, we established F1 hybrid ES cells derived from two sub-species of Mus musculus and established an in vitro neuronal differentiation system in which neuron-specific imprinting of Ube3a was recapitulated. With this system, we revealed that the switch from biallelic expression to maternal, monoallelic expression of Ube3a occurs late in neuronal development, during the neurite outgrowth period, and that the expression of endogenous antisense transcript from the Ube3a locus is up-regulated several hundred-fold during the same period. Our results suggest that evaluation of the quality of ES cells by studying their differentiation in vitro should include evaluation of epigenetic aspects, such as a comparison with the genomic imprinting status found in tissues in vivo, in addition to the evaluation of differentiation gene markers and morphology. Our F1 hybrid ES cells and in vitro differentiation system will allow researchers to investigate complex end-points such as neuron-specific genomic imprinting, and our F1 hybrid ES cells are a useful resource for other tissue-specific genomic imprinting and epigenetic analyses.

Identification of novel endogenous antisense transcripts by DNA microarray analysis targeting complementary strand of annotated genes

BackgroundRecent transcriptomic analyses in mammals have uncovered the widespread occurrence of endogenous antisense transcripts, termed natural antisense transcripts (NATs). NATs are transcribed from the opposite strand of the gene locus and are thought to control sense gene expression, but the mechanism of such regulation is as yet unknown. Although several thousand potential sense-antisense pairs have been identified in mammals, examples of functionally characterized NATs remain limited. To identify NAT candidates suitable for further functional analyses, we performed DNA microarray-based NAT screening using mouse adult normal tissues and mammary tumors to target not only the sense orientation but also the complementary strand of the annotated genes.ResultsFirst, we designed microarray probes to target the complementary strand of genes for which an antisense counterpart had been identified only in human public cDNA sources, but not in the mouse. We observed a prominent expression signal from 66.1% of 635 target genes, and 58 genes of these showed tissue-specific expression. Expression analyses of selected examples (Acaa1b and Aard) confirmed their dynamic transcription in vivo. Although interspecies conservation of NAT expression was previously investigated by the presence of cDNA sources in both species, our results suggest that there are more examples of human-mouse conserved NATs that could not be identified by cDNA sources. We also designed probes to target the complementary strand of well-characterized genes, including oncogenes, and compared the expression of these genes between mammary cancerous tissues and non-pathological tissues. We found that antisense expression of 95 genes of 404 well-annotated genes was markedly altered in tumor tissue compared with that in normal tissue and that 19 of these genes also exhibited changes in sense gene expression. These results highlight the importance of NAT expression in the regulation of cellular events and in pathological conditions.ConclusionOur microarray platform targeting the complementary strand of annotated genes successfully identified novel NATs that could not be identified by publically available cDNA data, and as such could not be detected by the usual "sense-targeting" microarray approach. Differentially expressed NATs monitored by this platform may provide candidates for investigations of gene function. An advantage of our microarray platform is that it can be applied to any genes and target samples of interest.

Control of alternative splicing through siRNA-mediated transcriptional gene silencing

When targeting promoter regions, small interfering RNAs (siRNAs) trigger a previously proposed pathway known as transcriptional gene silencing by promoting heterochromatin formation. Here we show that siRNAs targeting intronic or exonic sequences close to an alternative exon regulate the splicing of that exon. The effect occurred in hepatoma and HeLa cells with siRNA antisense strands designed to enter the silencing pathway, suggesting hybridization with nascent pre-mRNA. Unexpectedly, in HeLa cells the sense strands were also effective, suggesting that an endogenous antisense transcript, detectable in HeLa but not in hepatoma cells, acts as a target. The effect depends on Argonaute-1 and is counterbalanced by factors favoring chromatin opening or transcriptional elongation. The increase in heterochromatin marks (dimethylation at Lys9 and trimethylation at Lys27 of histone H3) at the target site, the need for the heterochromatin-associated protein HP1alpha and the reduction in RNA polymerase II processivity suggest a mechanism involving the kinetic coupling of transcription and alternative splicing.

Nkx2.2 antisense RNA overexpression enhanced oligodendrocytic differentiation

Nkx2.2AS is an endogenous antisense transcript to Nkx2.2 gene, subcellularly located in the cytoplasm. We tested if Nkx2.2AS transcripts have any biological function by regulating transcription level of Nkx2.2 coding gene in the differentiation of neural stem cells. Forced expression of Nkx2.2AS in neural stem cells enhanced induction of differentiation along lineage of oligodendrocytes. Further analyses showed that overexpression of Nkx2.2AS induced modest increase in Nkx2.2 mRNA level, suggesting that the upregulation of Nkx2.2 mRNA level can be a minor cause of enhanced induction of oligodendrocytic differentiation by Nkx2.2AS overexpression. These results together imply that Nkx2.2AS has a certain biological function which is likely to be not only based on the affect on transcription level of Nkx2.2 coding gene in cis, but also on the other mechanisms.

Naturally occurring antisense RNA of allatostatin gene in the prawn, Macrobrachium rosenbergii

Allatostatins are important regulatory neuropeptides which are widely distributed in invertebrates and execute their functions through either neural or humoral routes. However, the regulatory mechanism of the gene expression is unclear. In this paper, we report a naturally occurring antisense transcript, named as asMacro-AST A, of the crustacean FGLamide allatostatin gene (Macro-AST A) from the prawn, Macrobrachium rosenbergii. The asMacro-AST A contains an 843-bp sequence fully complementary to the 3′ end of the Macro-AST A. To our knowledge, this is the first report of a natural antisense transcript in crustacean and the first endogenous antisense transcript of all identified allatostatin genes. Northern blotting analysis demonstrated that the gene was expressed in the thoracic ganglia where the sense gene was also expressed. Furthermore, we have detected a RNA–RNA duplex between the sense–antisense complementary region by using RNase protection analysis and RT–PCR. These results suggest that the antisense gene may play a role in the regulation of Macro-AST A gene expression.

Natural antisense transcripts of Alzheimer's disease associated genes

Natural antisense transcripts (NATs), also named endogenous antisense transcripts, are a class of genes whose role in controlling gene expression is becoming more and more relevant. NATs might play important roles in gene expression and translation regulation. Present work investigated the presence of NATs of Alzheimer's disease associated genes including PRESENILIN1, PRESENILIN2, BACE1, BACE2, APP, APOE, TAU (MAPT), PRION, alpha-SYNUCLEIN (SNCA), NICASTRIN, PEN2, APH1A, APH1B as well as CD147 (BASIGIN), and the results revealed that APP, BACE2, APH1A, TAU, CD147 and alpha-SYNUCLEIN contain natural antisense transcripts. These NATs were characterized according to the sense-antisense overlapping information and potential functional mechanisms were proposed. Present findings provide preliminary but important information about transcription regulation of AD associated genes, which would further our understanding of the gene expression regulation of AD, and also suggest a novel potential strategy for the therapy of AD.

Non-coding RNA directed DNA demethylation of Sphk1 CpG island

The formation of DNA methylation patterns is one of the epigenetic events that underlie mammalian development. The Sphk1 CpG island is a target for tissue-dependent DNA methylation as well as a template for generating multiple subtypes. The number of mammalian non-coding RNA genes is rapidly expanding. In this study, we found endogenous antisense transcripts, Khps1 subtypes with different sizes (600–20,000nt). A subtype, Khps1a, was a 1290-bp, non-coding, 5′-capped and 3′-polyadenylated RNA that originated from the CpG island and overlapped with a tissue-dependent differentially methylated region (T-DMR) of Sphk1. Intriguingly, overexpression of two fragments of Khps1 caused demethylation of CG sites in the T-DMR. Furthermore, this RNA-directed demethylation was associated with DNA methylation at three CC(A/T)GG sites in the T-DMR. The link between the RNA-directed CG demethylation and non-CG methylation provides a novel mechanism of epigenetic regulation and potential tool for epigenetic manipulation of mammalian cells.

Molecular cloning and characterization of an endogenous antisense transcript of Nphs1

Mutations of NPHS1, the gene encoding the kidney glomerular filtration barrier protein nephrin, cause congenital nephrotic syndrome of the Finnish type. Nephrin is a component of the interpodocyte-spanning slit diaphragm: it mediates outside–in signaling and forms a nexus for homo- and heterotypic molecular interactions. When studying the nephrin-deficient mouse line generated by random insertional mutagenesis we unexpectedly discovered an endogenous antisense transcript originating from the nephrin-encoding locus. Further evidence of the antisense transcript (Nphs1as) was obtained by searching for Nphs1-like expressed sequence tags. Surprisingly, one clone showed exact complementarity in the antisense orientation. Nphs1as is expressed in the brain, thymus, and peripheral lymph nodes as well as in the embryonic stem cells. However, the mesenteric lymph nodes and the main sites of nephrin expression, the kidney and pancreas, were negative. Nphs1as is a continuous, polyadenylated mRNA that spans Nphs1 exons from 7 to 12 in the reverse orientation. The relative amounts of sense and antisense mRNAs as well as nephrin protein were determined by semiquantitative RT-PCR and immunoblotting, respectively, in various mouse tissues. These results suggest that Nphs1as may be important for the regulation of the appropriate tissue- and cell-type-specific expression of nephrin.

Regulation of the NPT gene by a naturally occurring antisense transcript.

The epithelial Na/Pi cotransporter (NaPi-II) is instrumental in maintaining phosphate (Pi) homeostasis in vertebrates. Hormones and metabolic factors (PTH, Pi availability) that acutely influence renal Pi excretion have been demonstrated to target NaPi-II expression. Upon stimulation, newly synthesized transporter molecules become integrated into the brush-border membrane to increase the Vmax of Pi uptake; reduction of Pi reabsorption is achieved by endocytosis of NaPi-II followed by lysosomal degradation of the protein. The long-term regulation of the protein is less well studied. Only recently, regulatory elements for vitamin D3 and Pi have been identified in the promoter region of the npt gene. However, signaling pathways leading to the activation of these regulatory sequences need to be established. Other reports suggested messenger RNA stability to play a role in the medium range regulation of NaPi-II expression. Recent findings in our laboratory added to the complex picture of npt gene regulation. We have identified npt-related endogenous antisense transcripts from mouse, zebrafish, and winter flounder. The two fish transcripts have been cloned and characterized; the mouse homolog has only very recently been detected. The transcripts are devoid of an open reading frame and appear in different splice forms. The evolutionary conservation of bidirectional transcription of the npt gene implies a regulatory function for the antisense transcript. In order to test the functional consequences of bidirectional transcription, we coexpressed sense and the antisense transcripts from zebrafish in Xenopus oocytes. Pi transport activity was reduced as a result of the presence of antisense RNA. Re-extraction of the RNA from injected oocytes followed by Northern blot revealed that the coexpression had no significant effect on the stability of either transcript. We concluded that the antisense mRNA interfered with the translation of the transporter if coexpressed in the Xenopus system. However, the regulatory mechanism(s) involving the npt-related antisense transcript is expected to be much more complicated in vivo, (i.e., requiring supplementary factors like double-stranded RNA recognizing proteins or specific RNases). It is planned to test this hypothesis by a transgenic zebrafish approach and/or knockout mice.

Duplications on human chromosome 22 reveal a novel Ret Finger Protein-like gene family with sense and endogenous antisense transcripts.

Analysis of 600 kb of sequence encompassing the beta-prime adaptin (BAM22) gene on human chromosome 22 revealed intrachromosomal duplications within 22q12-13 resulting in three active RFPL genes, two RFPL pseudogenes, and two pseudogenes of BAM22. The genomic sequence of BAM22vartheta1 shows a remarkable similarity to that of BAM22. The cDNA sequence comparison of RFPL1, RFPL2, and RFPL3 showed 95%-96% identity between the genes, which were most similar to the Ret Finger Protein gene from human chromosome 6. The sense RFPL transcripts encode proteins with the tripartite structure, composed of RING finger, coiled-coil, and B30-2 domains, which are characteristic of the RING-B30 family. Each of these domains are thought to mediate protein-protein interactions by promoting homo- or heterodimerization. The MID1 gene on Xp22 is also a member of the RING-B30 family and is mutated in Opitz syndrome (OS). The autosomal dominant form of OS shows linkage to 22q11-q12. We detected a polymorphic protein-truncating allele of RFPL1 in 8% of the population, which was not associated with the OS phenotype. We identified 6-kb and 1.2-kb noncoding antisense mRNAs of RFPL1S and RFPL3S antisense genes, respectively. The RFPL1S and RFPL3S genes cover substantial portions of their sense counterparts, which suggests that the function of RFPL1S and RFPL3S is a post-transcriptional regulation of the sense RFPL genes. We illustrate the role of intrachromosomal duplications in the generation of RFPL genes, which were created by a series of duplications and share an ancestor with the RING-B30 domain containing genes from the major histocompatibility complex region on human chromosome 6.

Making (anti)sense of non-coding sequence conservation.

A substantial fraction of vertebrate mRNAs contain long conserved blocks in their untranslated regions as well as long blocks without silent changes in their protein coding regions. These conserved blocks are largely comprised of unique sequence within the genome, leaving us with an important puzzle regarding their function. A large body of experimental data shows that these regions are associated with regulation of mRNA stability. Combining this information with the rapidly accumulating data on endogenous antisense transcripts, we propose that the conserved sequences form long perfect duplexes with antisense transcripts. The formation of such duplexes may be essential for recognition by post-transcriptional regulatory systems. The conservation may then be explained by selection against the dominant negative effect of allelic divergence.

Natural antisense transcripts of the S locus receptor kinase gene and related sequences in Brassica oleracea.

Gene expression can be inhibited by antisense RNA transcripts. Although this phenomenon is widely used to analyse gene function in plants, the molecular mechanisms involved are poorly understood. One approach to improving our understanding of antisense gene regulation is to analyse the function of endogenous antisense transcripts. To date, only a small number of plant genes have been shown to be transcribed in both directions and limited information is available concerning the role of natural antisense transcripts in plants. In this study, we have identified several natural antisense transcripts which hybridise to probes derived from the S locus receptor kinase gene (SRK). The RNase protection assay and reverse trancriptase-PCR were used to demonstrate that a proportion of the antisense transcripts are encoded directly by SRK. Using different RNase protection probes, regions of the promoter, exon I (which encodes the S domain) and intron I of SRK were shown to be transcribed in an antisense direction. An antisense SRK transcript was shown to inhibit translation of a sense transcript in vitro. The possible role of antisense SRK transcripts in vivo is discussed.

What makes an mRNA anti-sensi-itive?

Antisense RNA has been used for some time as a versatile tool for silencing gene expression. There is ample evidence for gene regulation by endogenous antisense transcripts in prokaryotes and increasing insight into the molecular mechanisms underlying such regulation. The introduction of antisense gene constructs into eukaryotes has now become routine but the mechanisms by which gene expression is inhibited are barely understood. In recent years, several examples of endogenous eukaryotic antisense transcripts have been discovered, some of which probably serve regulatory functions. Here we will discuss a model to explain mechanisms of antisense-mediated gene silencing.

Characterization of an endogenous RNA transcript with homology to the antisense strand of the human c-myc gene.

In addition to being regulated by a complex array of cis- and trans-acting factors, c-myc protooncogene expression may be modulated by antisense RNA transcripts. Our previous studies have determined that depletion of intracellular polyamines by alpha-difluoromethylornithine results in a marked decrease in the transcription of the human c-myc gene. Because of reports that antisense transcription occurs in the 5' and 3' regions of this gene, we used a genomic clone of the human c-myc gene to ascertain whether polyamine depletion might induce an antisense RNA transcript. These studies demonstrate that polyamine depletion of the human colon cancer cell line COLO 320 results in induction of an endogenous RNA transcript with high homology to the antisense strand of the second intervening sequence (PvuII-RsaI) of the c-myc gene. Furthermore, during such depletion, steady state levels of this transcript vary inversely to the sense direction c-myc RNA. RNase protection studies suggest that the antisense transcript may arise from a different gene locus than the c-myc gene. To further identify the origins of this RNA, a cDNA library was generated from size-selected RNA and screened with c-myc sequences. A 438-base pair cDNA was isolated with approximately 85% homology, to a 285-base region in the second intron of the c-myc gene. Computer homology analysis further reveals that a 120-base region within this cDNA also has approximately 85% homology to the antisense strands of a number of genes, including the growth-related genes, N-myc, p53, and thymidine kinase. These studies provide the initial characterization of an endogenous antisense RNA transcript which could influence cell growth by modulating the expression of c-myc and other genes.

N-myc mRNA forms an RNA-RNA duplex with endogenous antisense transcripts.

Nuclear runoff transcription studies revealed nearly equivalent sense and antisense transcription across exon 1 of the N-myc locus. Antisense primary transcription initiates at multiple sites in intron 1 and gives rise to stable polyadenylated and nonpolyadenylated transcripts. This pattern of antisense transcription, which is directed by RNA polymerase II, is independent of gene amplification and cell type. The nonpolyadenylated antisense transcripts have 5' ends which are complementary to the 5' ends of the N-myc sense mRNA. We determined, by using an RNase protection technique designed to detect in vivo duplexes, that most of the cytoplasmic nonpolyadenylated antisense RNA exists in an RNA-RNA duplex with approximately 5% of the sense N-myc mRNA. Duplex formation appeared to occur with only a subset of the multiple forms of the N-myc mRNA, with the precise transcriptional initiation site of the RNA playing a role in determining this selectivity. Cloning of each strand of the RNA-RNA duplex revealed that most duplexes included both exon 1 and intron 1 sequences, suggesting that duplex formation could modulate RNA processing by preserving a population of N-myc mRNA which retains intron 1.