Antisense Transcripts Research Articles

Modulation of sol mRNA expression by the long non-coding RNA Assolrna in Clostridium saccharoperbutylacetonicum affects solvent formation.

Solvents such as butanol are important platform chemicals and are often produced from petrochemical sources. Production of butanol and other compounds from renewable and sustainable resources can be achieved by solventogenic bacteria, such as the hyper-butanol producer Clostridium saccharoperbutylacetonicum. Its sol operon consists of the genes encoding butyraldehyde dehydrogenase, CoA transferase, and acetoacetate decarboxylase (bld, ctfA, ctfB, adc) and the gene products are involved in butanol and acetone formation. It is important to understand its regulation to further optimize the solvent production. In this study, a new long non-coding antisense transcript complementary to the complete sol operon, now called Assolrna, was identified by transcriptomic analysis and the regulatory mechanism of Assolrna was investigated. For this purpose, the promoter-exchange strain C. saccharoperbutylacetonicum ΔP asr ::P asr ** was constructed. Additionally, Assolrna was expressed plasmid-based under control of the native P asr promoter and the lactose-inducible P bgaL promoter in both the wild type and the promoter-exchange strain. Solvent formation was strongly decreased for all strains based on C. saccharoperbutylacetonicum ΔP asr ::P asr ** and growth could not be restored by plasmid-based complementation of the exchanged promoter. Interestingly, very little sol mRNA expression was detected in the strain C. saccharoperbutylacetonicum ΔP asr ::P asr ** lacking Assolrna expression. Butanol titers were further increased for the overexpression strain C. saccharoperbutylacetonicum [pMTL83151_asr_P bgaL ] compared to the wild type. These results suggest that Assolrna has a positive effect on sol operon expression. Therefore, a possible stabilization mechanism of the sol mRNA by Assolrna under physiological concentrations is proposed.

Long Noncoding RNA LEMD1-AS1 Increases LEMD1 Expression and Activates PI3K-AKT Pathway to Promote Metastasis in Oral Squamous Cell Carcinoma.

Background The survival rate of oral squamous cell carcinoma (OSCC) is only 50% due to a high incidence of metastasis. Long noncoding RNAs (lncRNAs) play a crucial role in OSCC genesis and progression, although their potential role in the metastasis of OSCC remains unclear. Methods The transcriptome of 5 metastatic and 5 nonmetastatic OSCC samples were assessed by RNA sequencing. The biological functions and regulatory mechanisms of LEMD1-AS1 in OSCC were explored by in vitro and in vivo assays. Results We identified 487 differentially expressed mRNAs (DEmRNAs) and 1507 differentially expressed lncRNAs (DElncRNAs) in OSCC with cervical lymph node (LN) metastasis relative to the nonmetastatic samples. In addition, both LEMD1-AS1 and its cognate LEMD1 were up-regulated in metastatic OSCC compared to nonmetastatic OSCC. Gain-of-function, loss-of-function, and rescue experiments indicated that LEMD1-AS1 upregulated LEMD1 to increase OSCC migration and invasion in vitro and in vivo. Mechanistically, LEMD1-AS1 stabilized LEMD1 and increased its mRNA and protein levels, and consequently activated the PI3K-AKT signaling pathway to facilitate OSCC metastasis. Conclusions We established the lncRNA-mRNA landscape of metastatic OSCC, which indicated that LEMD1-AS1 enhanced OSCC metastasis by stabilizing its antisense transcript LEMD1. Thus, LEMD1-AS1 is a potential biomarker for predicting metastasis, as well as a therapeutic target of OSCC.

RNA editing underlies genetic risk of common inflammatory diseases.

A major challenge in human genetics is to identify the molecular mechanisms of trait-associated and disease-associated variants. To achieve this, quantitative trait locus (QTL) mapping of genetic variants with intermediate molecular phenotypes such as gene expression and splicing have been widely adopted1,2. However, despite successes, the molecular basis for a considerable fraction of trait-associated and disease-associated variants remains unclear3,4. Here we show that ADAR-mediated adenosine-to-inosine RNA editing, a post-transcriptional event vital for suppressing cellular double-stranded RNA (dsRNA)-mediated innate immune interferon responses5-11, is an important potential mechanism underlying genetic variants associated with common inflammatory diseases. We identified and characterized 30,319 cis-RNA editing QTLs (edQTLs) across 49 human tissues. These edQTLs were significantly enriched in genome-wide association study signals for autoimmune and immune-mediated diseases. Colocalization analysis of edQTLs with disease risk loci further pinpointed key, putatively immunogenic dsRNAs formed by expected inverted repeat Alu elements as well as unexpected, highly over-represented cis-natural antisense transcripts. Furthermore, inflammatory disease risk variants, in aggregate, were associated with reduced editing of nearby dsRNAs and induced interferon responses in inflammatory diseases. This unique directional effect agrees with the established mechanism that lack of RNA editing by ADAR1 leads to the specific activation of the dsRNA sensor MDA5 and subsequent interferon responses and inflammation7-9. Our findings implicate cellular dsRNA editing and sensing as a previously underappreciated mechanism of common inflammatory diseases.

Mechanism of miR-338-3p in sepsis-induced acute lung injury via indirectly modulating ATF4

Sepsis is recognized as an inflammation-related syndrome in response to invading pathogens. Many patients suffer from sepsis including transplant recipients. Lipopolysaccharide (LPS) is known to trigger sepsis-related organ dysfunction. This study expounded on the possible effect of microRNA (miR)-338-3p in sepsis-induced acute lung injury (ALI). Firstly, human bronchial epithelial cell line 16HBE received LPS treatment to establish the cell models of sepsis-induced ALI. The expression patterns of miR-338-3p, long non-coding RNA OPA-interacting protein 5 antisense transcript 1 (lncRNA OIP5-AS1), and activating transcription factor 4 (ATF4) in 16HBE cells were examined. Afterwards, 16HBE cell viability, the apoptosis rate, and the levels of inflammation and lactate dehydrogenase (LDH) were determined to assess the degree of cell injury. We disclosed that LPS treatment triggered 16HBE cell injury, downregulated miR-338-3p, and upregulated OIP5-AS1 and ATF4. miR-338-3p overexpression repressed LPS-induced 16HBE cell injury. miR-338-3p diminished OIP5-AS1 stability via binding to OIP5-AS1 and downregulated OIP5-AS1 expression and OIP5-AS1 can enhance ATF4 mRNA stability and upregulate ATF4 mRNA level. The rescue experiments showed that ATF4 overexpression aggravated LPS-induced 16HBE cell injury. Overall, miR-338-3p overexpression decreased OIP5-AS1 expression and stability and further downregulated ATF4 mRNA level, thereby mitigating LPS-induced 16HBE cell injury.

HTLV-1 intragenic viral enhancer influences immortalization phenotype in vitro, but is dispensable for persistence and disease development in animal models.

Human T-cell leukemia virus type 1 (HTLV-1) is the causative infectious agent of adult T-cell leukemia/lymphoma (ATL) and chronic neurological disease. The disparity between silenced sense transcription versus constitutively active antisense (Hbz) transcription from the integrated provirus is not fully understood. The presence of an internal viral enhancer has recently been discovered in the Tax gene near the 3’ long terminal repeat (LTR) of HTLV-1. In vitro, this enhancer has been shown to bind SRF and ELK-1 host transcription factors, maintain chromatin openness and viral gene transcription, and induce aberrant host gene transcription near viral integration sites. However, the function of the viral enhancer in the context of early HTLV-1 infection events remains unknown. In this study, we generated a mutant Enhancer virus (mEnhancer) and evaluated its effects on HTLV-1-mediated in vitro immortalization, establishment of persistent infection with an in vivo rabbit model, and disease development in a humanized immune system (HIS) mouse model. The mEnhancer virus was able to establish persistent infection in rabbits, and there were no significant differences in proviral load or HTLV-1-specific antibody responses over a 25-week study. However, rabbits infected with the mEnhancer virus had significantly decreased sense and antisense viral gene expression at 12-weeks post-infection. HIS mice infected with wt or mEnhancer virus showed similar disease progression, proviral load, and viral gene expression. While mEnhancer virus was able to sufficiently immortalize primary T-lymphocytes in cell culture, the immortalized cells had an altered phenotype (CD8+ T-cells), decreased proviral load, decreased sense and anti-sense gene expression, and altered cell cycle progression compared to HTLV-1.wt immortalized cells (CD4+ T-cells). These results suggest that the HTLV-1 enhancer element alone does not determine persistence or disease development but plays a pivotal role in regulating viral gene expression.

A Novel ATM Antisense Transcript ATM-AS Positively Regulates ATM Expression in Normal and Breast Cancer Cells

ObjectiveThe ataxia telangiectasia mutated (ATM) gene is a master regulator in cellular DNA damage response. The dysregulation of ATM expression is frequent in breast cancer, and is known to be involved in the carcinogenesis and prognosis of cancer. However, the underlying mechanism remains unclear. The bioinformatic analysis predicted a potential antisense transcript ATM-antisense (AS) from the opposite strand of the ATM gene. The purpose of this study was to identify ATM-AS and investigate the possible effect of ATM-AS on the ATM gene regulation.MethodsSingle strand-specific RT-PCR was performed to verify the predicted antisense transcript ATM-AS within the ATM gene locus. qRT-PCR and Western blotting were used to detect the expression levels of ATM-AS and ATM in normal and breast cancer cell lines as well as in tissue samples. Luciferase reporter gene assays, biological mass spectrometry, ChIP-qPCR and RIP were used to explore the function of ATM-AS in regulating the ATM expression. Immunofluorescence and host-cell reactivation (HCR) assay were performed to evaluate the biological significance of ATM-AS in ATM-mediated DNA damage repair. Breast cancer tissue samples were used for evaluating the correlation of the ATM-AS level with the ATM expression as well as prognosis of the patients.ResultsThe ATM-AS significantly upregulated the ATM gene activity by recruiting KAT5 histone acetyltransferase to the gene promoter. The reduced ATM-AS level led to the abnormal downregulation of ATM expression, and impaired the ATM-mediated DNA damage repair in normal breast cells in vitro. The ATM-AS level was positively correlated with the ATM expression in the examined breast cancer tissue samples, and the patient prognosis.ConclusionThe present study demonstrated that ATM-AS, an antisense transcript located within the ATM gene body, is an essential positive regulator of ATM expression, and functions by mediating the binding of KAT5 to the ATM promoter. These findings uncover the novel mechanism underlying the dysregulation of the ATM gene in breast cancer, and enrich our understanding of how an antisense transcript regulates its host gene.

GGC Repeat Expansion of RILPL1 is Associated with Oculopharyngodistal Myopathy.

Oculopharyngodistal myopathy (OPDM) is an adult-onset neuromuscular disease characterized by progressive ptosis, dysarthria, ophthalmoplegia, and distal muscle weakness. Recent studies revealed that GGC repeat expansions in 5'-UTR of LRP12, GIPC1, and NOTCH2NLC are associated with OPDM. Despite these advances, approximately 30% of OPDM patients remain genetically undiagnosed. Herein, we aim to investigate the genetic basis for undiagnosed OPDM patients in two unrelated Chinese Han families. Parametric linkage analysis was performed. Long-read sequencing followed by repeat-primed polymerase chain reaction and amplicon length polymerase chain reaction were used to determine the genetic cause. Targeted methylation sequencing was implemented to detect epigenetic changes. The possible pathogenesis mechanism was investigated by quantitative polymerase chain reaction, immunoblotting, RNA fluorescence in situ hybridization, and immunofluorescence staining of muscle biopsy samples. The disease locus was mapped to 12q24.3. Subsequently, GGC repeat expansion in the promoter region of RILPL1 was identified in six OPDM patients from two families, findings consistent with a founder effect, designated as OPDM type4. Targeted methylation sequencing revealed hypermethylation at the RILPL1 locus in unaffected individuals with ultralong expansion. Analysis of muscle samples showed no significant differences in RILPL1 mRNA or RILPL1 protein levels between patients and controls. Public CAGE-seq data indicated that alternative transcription start sites exist upstream of the RefSeq-annotated RILPL1 transcription start site. Strand-specific RNA-seq data revealed bidirectional transcription from the RILPL1 locus. Finally, fluorescence in situ hybridization/immunofluorescence staining showed that both sense and antisense transcripts formed RNA foci, and were co-localized with hnRNPA2B1 and p62 in the intranuclear inclusions of OPDM type4 patients. Our findings implicate abnormal GGC repeat expansions in the promoter region of RILPL1 as a novel genetic cause for OPDM, and suggest a methylation mechanism and a potential RNA toxicity mechanism are involved in OPDM type4 pathogenesis. ANN NEUROL 2022;92:512-526.

HOTAIR interacts with PRC2 complex regulating the regional preadipocyte transcriptome and human fat distribution.

Mechanisms governing regional human adipose tissue (AT) development remain undefined. Here, we show that the long non-coding RNA HOTAIR (HOX transcript antisense RNA) is exclusively expressed in gluteofemoral AT, where it is essential for adipocyte development. We find that HOTAIR interacts with polycomb repressive complex 2 (PRC2) and we identify core HOTAIR-PRC2 target genes involved in adipocyte lineage determination. Repression of target genes coincides with PRC2 promoter occupancy and H3K27 trimethylation. HOTAIR is also involved in modifying the gluteal adipocyte transcriptome through alternative splicing. Gluteal-specific expression of HOTAIR is maintained by defined regions of open chromatin across the HOTAIR promoter. HOTAIR expression levels can be modified by hormonal (estrogen, glucocorticoids) and genetic variation (rs1443512 is a HOTAIR eQTL associated with reduced gynoid fat mass). These data identify HOTAIR as a dynamic regulator of the gluteal adipocyte transcriptome and epigenome with functional importance for human regional AT development.

Long non-coding RNA opa-interacting protein 5 antisense transcript 1 (LncRNA OIP5-AS1) promoted cisplatin resistance in nasopharyngeal carcinoma via the miR-378a-3p/nicotinamide N-methyltransferase axis

LncRNAs involve in chemoresistance of human cancers. However, the role and molecular mechanisms of lncRNA OIP5-AS1 in the chemoresistance of NPC are still unexplored. In our study, upregulated OIP5-AS1 was found in cisplatin (CDDP)-resistant NPC tumors and cell lines. Functional assays revealed OIP5-AS1 knockdown suppressed malignant behaviors, but stimulated apoptosis of CDDP-resistant NPC cells. Furthermore, we demonstrated OIP5-AS1 positively regulated NNMT by directly targeting miR-378a-3p. In addition, its inhibition partially abolished the inhibitory effects of OIP5-AS1 silencing on malignancy of CDDPresistant NPC cells, whereas NNMT knockdown reverse these effects. In sum, our results indicated OIP5-AS1 contributed to the CDDP resistance of NPC by sponging miR-378a-3p to increase NNMT expression.

Abstract 5828: Functional role of long non-coding RNA YTHDF3-AS1 in breast cancer

Abstract Breast cancer (BrCa) is the most common form of cancer in women worldwide, impacting nearly 2.1 million women each year. Importantly, current treatment options for metastatic breast cancer fail to elicit an anti-tumor response, leading to a 5-year survival rate below 30%. While most classes of therapy focus on inhibiting or targeting specific proteins, long non-coding RNAs (lncRNAs) offer a unique, relatively untapped, source of potential therapeutic targets. Ongoing experiments are evaluating the regulatory role of the lncRNA YTHDF3-AS1 (ENSG00000270673), which has the potential to develop into a novel therapeutic intervention of BrCa. Through genome-wide analysis, we found that YTHDF3-AS1 expression correlates with essential developmental regulatory genes such as CA3, VPS28, EXOSC4, and TM2D2 and according to pathway analysis is likely linked with the Wnt pathway. Additionally, TCGA data reveals that over 5% of patients with BrCa have an increased copy number of YTHDF3-AS1. Although the role of YTHDF3-AS1 has yet to be explored, YTHDF3 expression was recently shown to be a critical driver of breast cancer metastasis. Of note, YTHDF3-AS1 is upstream of the protein-coding region, but within the promoter region of the YTHDF3 gene. Mechanistically, this allows the lncRNA YTHDF3-AS1 to potentially upregulate the expression of the protein-coding YTHDF3 gene, as two known functions of antisense lncRNAs describe the ability to 1) induce nearby gene expression through binding to the promoter region and 2) stabilize the RNA product of the nearby gene through binding to the 5’UTR. Interestingly, this manner of relationship between antisense (or "divergent") lncRNAs and their protein-coding partner was shown to be highly prevalent in pluripotent stem cells, with the downregulation of both the antisense and protein-coding transcripts following differentiation. Together, our preliminary analysis and previous literature suggest that the lncRNA YTHDF3-AS1 may contribute to the upregulation of YTHDF3, which is a known metastatic promoting protein. Our current study aims to determine the functional role of YTHDF3-AS1 in BrCa oncogenesis, with an emphasis on the mechanistic actions of this lncRNA for the development of future therapeutic approaches. Citation Format: Alakesh Bera, Surya Radhakrishnan, Eric Russ, Diya Biswas, Madhan Subramanian, Harvey B. Pollard, Hai Hu, Craig D. Shriver, Roopa Biswas, Meera Srivastava. Functional role of long non-coding RNA YTHDF3-AS1 in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5828.

Native RNA sequencing in fission yeast reveals frequent alternative splicing isoforms.

The unicellular yeast Schizosaccharomyces pombe (fission yeast) retains many of the splicing features observed in humans and is thus an excellent model to study the basic mechanisms of splicing. Nearly half the genes contain introns, but the impact of alternative splicing in gene regulation and proteome diversification remains largely unexplored. Here we leverage Oxford Nanopore Technologies native RNA sequencing (dRNA), as well as ribosome profiling data, to uncover the full range of polyadenylated transcripts and translated open reading frames. We identify 332 alternative isoforms affecting the coding sequences of 262 different genes, 97 of which occur at frequencies >20%, indicating that functional alternative splicing in S. pombe is more prevalent than previously suspected. Intron retention events make ∼80% of the cases; these events may be involved in the regulation of gene expression and, in some cases, generate novel protein isoforms, as supported by ribosome profiling data in 18 of the intron retention isoforms. One example is the rpl22 gene, in which intron retention is associated with the translation of a protein of only 13 amino acids. We also find that lowly expressed transcripts tend to have longer poly(A) tails than highly expressed transcripts, highlighting an interdependence between poly(A) tail length and transcript expression level. Finally, we discover 214 novel transcripts that are not annotated, including 158 antisense transcripts, some of which also show translation evidence. The methodologies described in this work open new opportunities to study the regulation of splicing in a simple eukaryotic model.

MiR398b and AtC2GnT form a negative feedback loop to regulate Arabidopsis thaliana resistance against Phytophthora parasitica.

Oomycetes are diploid eukaryotic microorganisms that seriously threaten sustainable crop production. MicroRNAs (miRNAs) and corresponding natural antisense transcripts (NATs) are important regulators of multiple biological processes. However, little is known about their roles in plant immunity against oomycete pathogens. In this study, we report the identification and functional characterization of miR398b and its cis-NAT, the core-2/I-branching beta-1,6-N-acetylglucosaminyltransferase gene (AtC2GnT), in plant immunity. Gain- and loss-of-function assays revealed that miR398b mediates Arabidopsis thaliana susceptibility to Phytophthora parasitica by targeting Cu/Zn-Superoxidase Dismutase1 (CSD1) and CSD2, leading to suppressed expression of CSD1 and CSD2 and decreased plant disease resistance. We further showed that AtC2GnT transcripts could inhibit the miR398b-CSDs module via inhibition of pri-miR398b expression, leading to elevated plant resistance to P. parasitica. Furthermore, quantitative reverse transcription PCR, RNA ligase-mediated 5'-amplification of cDNA ends (RLM-5' RACE), and transient expression assays indicated that miR398b suppresses the expression of AtC2GnT. We generated AtC2GnT-silenced A. thaliana plants by CRISPR/Cas9 or RNA interference methods, and the Nicotiana benthamiana NbC2GnT-silenced plants by virus-induced gene silencing. Pathogenicity assays showed that the C2GnT-silenced plants were more susceptible, while AtC2GnT-overexpressing plants exhibited elevated resistance to P. parasitica. AtC2GnT encodes a Golgi-localized protein, and transient expression of AtC2GnT enhanced N. benthamiana resistance to Phytophthora pathogens. Taken together, our results revealed a positive role of AtC2GnT and a negative regulatory loop formed by miR398b and AtC2GnT in regulating plant resistance to P. parasitica.

Antisense transcription from lentiviral gene targeting linked to an integrated stress response in colorectal cancer cells

Advances in gene therapy research have resulted in the successful development of new therapies for clinical use. Here, we explored a gene targeting approach to deplete ephrinB2 from colorectal cancer cells using an inducible lentiviral vector. EphrinB2, a transmembrane ephrin ligand, promotes colorectal cancer cell growth and viability and predicts poor patient survival when expressed at high levels in colorectal cancer tissues. We discovered that lentiviral vector integration and expression in the host DNA frequently drive divergent host gene transcription, generating antisense reads coupled with splicing events and generation of chimeric vector/host transcripts. Antisense transcription of host DNA was linked to development of an integrated stress response and cell death. Despite recent successes, off-target effects remain a concern in genetic medicine. Our results provide evidence that divergent gene transcription is a previously unrecognized off-target effect of lentiviral vector integration with built-in properties for regulation of gene expression.

Transcription elongation is finely tuned by dozens of regulatory factors.

Understanding the complex network that regulates transcription elongation requires the quantitative analysis of RNA polymerase II (Pol II) activity in a wide variety of regulatory environments. We performed native elongating transcript sequencing (NET-seq) in 41 strains of Saccharomyces cerevisiae lacking known elongation regulators, including RNA processing factors, transcription elongation factors, chromatin modifiers, and remodelers. We found that the opposing effects of these factors balance transcription elongation and antisense transcription. Different sets of factors tightly regulate Pol II progression across gene bodies so that Pol II density peaks at key points of RNA processing. These regulators control where Pol II pauses with each obscuring large numbers of potential pause sites that are primarily determined by DNA sequence and shape. Antisense transcription varies highly across the regulatory landscapes analyzed, but antisense transcription in itself does not affect sense transcription at the same locus. Our findings collectively show that a diverse array of factors regulate transcription elongation by precisely balancing Pol II activity.

Identification and characterization of a novel enhancer in the HTLV-1 proviral genome

Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus that causes adult T-cell leukemia/lymphoma (ATL), a cancer of infected CD4+ T-cells. There is both sense and antisense transcription from the integrated provirus. Sense transcription tends to be suppressed, but antisense transcription is constitutively active. Various efforts have been made to elucidate the regulatory mechanism of HTLV-1 provirus for several decades; however, it remains unknown how HTLV-1 antisense transcription is maintained. Here, using proviral DNA-capture sequencing, we found a previously unidentified viral enhancer in the middle of the HTLV-1 provirus. The transcription factors, SRF and ELK-1, play a pivotal role in the activity of this enhancer. Aberrant transcription of genes in the proximity of integration sites was observed in freshly isolated ATL cells. This finding resolves certain long-standing questions concerning HTLV-1 persistence and pathogenesis. We anticipate that the DNA-capture-seq approach can be applied to analyze the regulatory mechanisms of other oncogenic viruses integrated into the host cellular genome.

LncRNA HDAC11-AS1 Suppresses Atherosclerosis by Inhibiting HDAC11-Mediated Adropin Histone Deacetylation.

LncRNA HDAC11-AS1 (HDAC11-AS1) is the natural antisense transcript of HDAC11, a key enzyme for DNA histone deacetylation. We evaluated the role of HDAC11-AS1 in atherosclerosis. In this research, we found that HDAC11-AS1 ameliorated blood lipid levels and atherosclerosis in high fat-dieted apoE-/- mice by regulating HDAC11 negatively. The change in blood lipid levels is related to the expression of LPL, which is enhanced by HDAC11-AS1 through regulating adropin histone deacetylation in vitro and in vivo. In conclusion, HDAC11-AS1 plays an anti-atherogenic role through adropin to induce LPL expressions, thereby enhancing TG metabolism. The results are valuable for the further development of HDAC11-AS1 and its clinical applications. It provides a new clinical therapeutic target for cardiovascular disease treatment.

Functional and Pathogenic Roles of Retroviral Antisense Transcripts.

Exogenous retroviruses such as human immunodeficiency virus type 1 (HIV-1), human T-cell leukemia virus type 1 (HTLV-1) and bovine leukemia virus (BLV) can cause various diseases including immunodeficiency, inflammatory diseases and hematologic malignancies. These retroviruses persistently infect their hosts. Therefore, they need to evade host immune surveillance. One way in which these viruses might avoid immune detection is to utilize functional RNAs, rather than proteins, for certain activities, because RNAs are not recognized by the host immune system. HTLV-1 encodes the HTLV-1 bZIP factor (HBZ) gene in the antisense strand of the provirus. The HBZ protein is constantly expressed in HTLV-1 carriers and patients with adult T-cell leukemia-lymphoma, and it plays critical roles in pathogenesis. However, HBZ not only encodes this protein, but also functions as mRNA. Thus, HBZ gene mRNA is bifunctional. HIV-1 and BLV also encode long non-coding RNAs as antisense transcripts. In this review, we reshape our current understanding of how these antisense transcripts function and how they influence disease pathogenesis.

LncRNA EGFR-AS1 facilitates leiomyosarcoma progression and immune escape via the EGFR-MYC-PD-L1 axis.

this study aimed to investigate the role of long non-coding RNA (lncRNA) epidermal growth factor receptor antisense RNA 1 (EGFR-AS1), an antisense transcript of EGFR, in leiomyosarcoma (LMS) and the underlying mechanisms. levels of EGFR-AS1 and programmed death ligand 1 (PD-L1) were measured in LMS tissues and cell lines using quantitative real-time PCR (qRT-PCR), as well as western blotting and/or immunohistochemical staining; flow cytometry was employed to validate the role of EGFR-AS1 in altering the activity of CD8+ T cells; interaction of EGFR-AS1 and EGFR was determined by fluorescent in situ hybridization (FISH) and RNA pull-down; regulation of MYC on the PD-L1 promoter was assessed by chromatin immunoprecipitation (ChIP); a xenograft in vivo tumor growth assay was applied to verify the EGFR-AS1/EGFR/MYC/PD-L1 axis in vivo. up-regulation of EGFR-AS1 and PD-L1 in LMS tissues was negatively correlated with CD8+ T-cell infiltration; EGFR-AS1 positively regulated PD-L1, thereby strengthening interaction of LMS cells and CD8+ T cells and triggering CD8+ T cell apoptosis via the PD-1/PD-L1 checkpoint; EGFR-AS1 co-localized and interacted with EGFR to promote MYC activity; MYC was identified as a transcriptional activator of PD-L1. lncRNA EGFR-AS1 was demonstrated to increase PD-L1 expression through the EGFR/MYC pathway in LMS cells, thereby repressing T-cell infiltration and contributing to immune escape.

Antisense-mediated repression of SAGA-dependent genes involves the HIR histone chaperone.

Eukaryotic genomes are pervasively transcribed by RNA polymerase II (RNAPII), and transcription of long non-coding RNAs often overlaps with coding gene promoters. This might lead to coding gene repression in a process named Transcription Interference (TI). In Saccharomyces cerevisiae, TI is mainly driven by antisense non-coding transcription and occurs through re-shaping of promoter Nucleosome-Depleted Regions (NDRs). In this study, we developed a genetic screen to identify new players involved in Antisense-Mediated Transcription Interference (AMTI). Among the candidates, we found the HIR histone chaperone complex known to be involved in de novo histone deposition. Using genome-wide approaches, we reveal that HIR-dependent histone deposition represses the promoters of SAGA-dependent genes via antisense non-coding transcription. However, while antisense transcription is enriched at promoters of SAGA-dependent genes, this feature is not sufficient to define the mode of gene regulation. We further show that the balance between HIR-dependent nucleosome incorporation and transcription factor binding at promoters directs transcription into a SAGA- or TFIID-dependent regulation. This study sheds light on a new connection between antisense non-coding transcription and the nature of coding transcription initiation.

Epimutations in both the TESK2 and MMACHC promoters in the Epi-cblC inherited disorder of intracellular metabolism of vitamin B12

Backgroundepi-cblC is a recently discovered inherited disorder of intracellular vitamin B12 metabolism associating hematological, neurological, and cardiometabolic outcomes. It is produced by an epimutation at the promoter common to CCDC163P and MMACHC, which results from an aberrant antisense transcription due to splicing mutations in the antisense PRDX1 gene neighboring MMACHC. We studied whether the aberrant transcription produced a second epimutation by encompassing the CpG island of the TESK2 gene neighboring CCDC163P.MethodsWe unraveled the methylome architecture of the CCDC163P–MMACHC CpG island (CpG:33) and the TESK2 CpG island (CpG:51) of 17 epi-cblC cases. We performed an integrative analysis of the DNA methylome profiling, transcriptome reconstruction of RNA-sequencing (RNA-seq), chromatin immunoprecipitation sequencing (ChIP-Seq) of histone H3, and transcription expression of MMACHC and TESK2.ResultsThe PRDX1 splice mutations and activation of numerous cryptic splice sites produced antisense readthrough transcripts encompassing the bidirectional MMACHC/CCDC163P promoter and the TESK2 promoter, resulting in the silencing of both the MMACHC and TESK2 genes through the deposition of SETD2-dependent H3K36me3 marks and the generation of epimutations in the CpG islands of the two promoters.ConclusionsThe antisense readthrough transcription of the mutated PRDX1 produces an epigenetic silencing of MMACHC and TESK2. We propose using the term 'epi-digenism' to define this epigenetic disorder that affects two genes. Epi-cblC is an entity that differs from cblC. Indeed, the PRDX1 and TESK2 altered expressions are observed in epi-cblC but not in cblC, suggesting further evaluating the potential consequences on cancer risk and spermatogenesis.