Antisense Transcripts Research Articles

Cotranscriptional splicing is required in the cold to produce COOLAIR isoforms that repress Arabidopsis FLC

Plants use seasonal cold to time the transition to reproductive development. Short- and long-term cold exposure is registered via parallel transcriptional shutdown and Polycomb-dependent epigenetic silencing of the Arabidopsis thaliana major flowering repressor locus FLOWERING LOCUS C (FLC). The cold-induced antisense transcripts (COOLAIR) determine the dynamics of FLC transcriptional shutdown, but the thermosensory mechanisms are still unresolved. Here, through a forward genetic screen, we identify a mutation that perturbs cold-induced COOLAIR expression and FLC repression. The mutation is a hypomorphic allele of SUPPRESSORS OF MEC-8 AND UNC-52 1 (SMU1), a conserved subunit of the spliceosomal B complex. SMU1 interacts in vivo with the proximal region of nascent COOLAIR and RNA 3' processing/cotranscriptional regulators and enhances COOLAIR proximal intron splicing to promote specific COOLAIR isoforms. SMU1 also interacts with ELF7, an RNA Polymerase II Associated Factor (Paf1) component and limits COOLAIR transcription. Cold thus changes cotranscriptional splicing/RNA Pol II functionality in an SMU1-dependent mechanism to promote two different isoforms of COOLAIR that lead to reduced FLC transcription. Such cotranscriptional mechanisms are emerging as important regulators underlying plasticity in gene expression.

Reduction of antisense transcription affects bovine leukemia virus replication and oncogenesis.

In sheep infected with bovine leukemia virus (BLV), transcription of structural, enzymatic, and accessory genes is silenced. However, the BLV provirus transcribes a series of non-coding RNAs that remain undetected by the host immune response. Specifically, three RNAs (AS1-L, AS1-S, and AS2) are consistently expressed from the antisense strand, originating from transcriptional initiation at the 3'-Long Terminal Repeat (LTR). To investigate the role of these non-coding RNAs in viral replication and pathogenesis, a reverse genetics approach was devised, capitalizing on a mechanistic disparity in transcription initiation between the 5' and 3' promoters. A two-nucleotide mutation (GG>TA) in the TFIIB-recognition element (BRE) impaired antisense transcription originating from the 3'-LTR. In the context of the provirus, this 2bp mutation significantly diminished the expression of antisense RNAs, while not notably affecting sense transcription. When inoculated to sheep, the mutated provirus was infectious but exhibited reduced replication levels, shedding light on the role of antisense transcription in vivo. In comparison to lymphoid organs in sheep infected with a wild-type (WT) provirus, the mutant demonstrated alterations in both the spatial distribution and rates of cell proliferation in the lymph nodes and the spleen. Analysis through RNA sequencing and RT-qPCR unveiled an upregulation of the Hmcn1/hemicentin-1 gene in B-lymphocytes from sheep infected with the mutated provirus. Further examination via confocal microscopy and immunohistochemistry revealed an increase in the amount of hemicentin-1 protein encoded by Hmcn1 in peripheral blood mononuclear cells (PBMCs) and lymphoid organs of sheep infected with the mutant. RNA interference targeting Hmcn1 expression impacted the migration of ovine kidney (OVK) cells in vitro. In contrast to the WT, the mutated provirus showed reduced oncogenicity when inoculated into sheep. Collectively, this study underscores the essential role of antisense transcription in BLV replication and pathogenicity. These findings may offer valuable insights into understanding the relevance of antisense transcription in the context of human T-cell leukemia virus (HTLV-1).

Spt5 orchestrates cryptic transcript suppression and transcriptional directionality

Spt5 is a well-conserved factor that manipulates multiple stages of transcription from promoter-proximal pausing (PPP) to termination. Recent studies have revealed an unexpected increase of antisense transcripts near promoters in cells expressing mutant Spt5. Here, we identify Spt5p-restricted intragenic antisense transcripts and their close relationship with sense transcription in yeast. We confirm that Spt5 CTR phosphorylation is also important to retain Spt5’s facility to regulate antisense transcription. The genes whose antisense transcription is strongly suppressed by Spt5p share strong endogenous sense transcription and weak antisense transcription, and this pattern is conserved in humans. Mechanistically, we found that Spt5p depletion increased histone acetylation to initiate intragenic antisense transcription by altering chromatin structure. We additionally identified termination factors that appear to be involved in the ability of Spt5p to restrict antisense transcription. By unveiling a new role of Spt5 in finely balancing the bidirectionality of transcription, we demonstrate that Spt5-mediated suppression of DSIF complex regulated-unstable transcripts (DUTs) is essential to sustain the accurate transcription by RNA polymerase II.

Long-read transcriptomics of Ostreid herpesvirus 1 uncovers a conserved expression strategy for the capsid maturation module and pinpoints a mechanism for evasion of the ADAR-based antiviral defence

Abstract Ostreid herpesvirus 1 (OsHV-1), a member of the family Malacoherpesviridae (order Herpesvirales), is a major pathogen of bivalves. However, the molecular details of the malacoherpesvirus infection cycle and its overall similarity to the replication of mammalian herpesviruses (family Orthoherpesviridae) remain obscure. Here, to gain insights into the OsHV-1 biology, we performed long read sequencing of infected blood clams, Anadara broughtonii, which yielded over one million OsHV-1 long reads. This data enabled the annotation of the viral genome with 78 gene units and 274 transcripts, of which 67 were polycistronic mRNAs, 35 ncRNAs and 20 natural antisense transcripts (NATs). Transcriptomics and proteomics data indicate preferential transcription and independent translation of the capsid scaffold protein as an OsHV-1 capsid maturation protease isoform. The conservation of this transcriptional architecture across Herpesvirales likely indicates its functional importance and ancient origin. Moreover, we traced RNA editing events using short read sequencing and supported the presence of inosine nucleotides in native OsHV-1 RNA, consistent with the activity of ADAR1. Our data suggests that, whereas RNA hyper-editing is concentrated in specific regions of the OsHV-1 genome, single nucleotide editing is more dispersed along the OsHV-1 transcripts. In conclusion, we reveal the existence of a conserved pan-Herpesvirales transcriptomic architecture of the capsid maturation module and uncover a transcription-based viral counter defence mechanism, which presumably facilitates the evasion of the host ADAR antiviral system.

Impacts of the feedback loop between sense-antisense RNAs in regulating circadian rhythms

Antisense transcripts are a unique group of non-coding RNAs and play regulatory roles in a variety of biological processes, including circadian rhythms. Per2AS is an antisense transcript to the sense core clock gene Period2 (Per2) in mouse and its expression is rhythmic and antiphasic to Per2. To understand the impact of Per2AS-Per2 interaction, we developed a new mathematical model that mechanistically described the mutually repressive relationship between Per2 and Per2AS. This mutual repression can regulate both amplitude and period of circadian oscillation by affecting a negative feedback regulation of Per2. Simulations from this model also fit with experimental observations that could not be fully explained by our previous model. Our revised model can not only serve as a foundation to build more detailed models to better understand the impact of Per2AS-Per2 interaction in the future, but also be used to analyze other sense-antisense RNA pairs that mutually repress each other.

Association between human telomerase reverse transcriptase (hTERT) MNS16A polymorphism and risk of breast cancer.

It is well known that telomerase activity is suppressed in normal human tissues and reactivated in tumors, suggesting that the human telomerase reverse transcriptase (hTERT, MIM: 187270) gene may be involved in carcinogenesis. A polymorphic tandem repeat minisatellite located downstream of exon 16 of hTERT and upstream in the putative promoter region of an antisense hTERT transcript, termed MNS16A, results in a functional polymorphism. Because the association between the MNS16A genetic polymorphism and breast cancer (BC) risk remains an open question, the present case-control study was conducted in Shiraz (Fars Province, Southern Iran). A total of 711 samples were collected, including 362 BC patients and 349 healthy individuals. Genotyping was performed by polymerase chain reaction method. Alleles were determined by classifying DNA amplicons of less than and greater than 300bp as short (S) and long (L) alleles, respectively. Different inheritance models (codominant, dominant, recessive, overdominant genotype models and the allele model) were used to evaluate the association between the MNS16A polymorphism and the risk of BC. No significant association was observed in any of the analyses. It should be noted that the statistical power of the comparisons was low. The present study did not support the association between hTERT MNS16A polymorphism and breast cancer risk. Similar studies in other populations with larger sample sizes are needed to determine the association between the hTERT MNS16A polymorphism and susceptibility to breast cancer.

FOXN3-AS1: A Candidate Prognostic Marker and Epigenetic Target with Immunotherapeutic Implications in Acute Myeloid Leukemia.

We focused on the FOXN3 gene and selected its antisense transcripts (FOXN3-AS1) to investigate its potential involvement in acute myeloid leukemia (AML). Several integrated multi-omics datasets have expanded the horizons of the cancer landscape. With the emergence of new high-throughput technologies, a large number of non-coding RNAs have been confirmed to be involved in the pathogenesis of different types of hematological malignancies. We conducted experimental validation using quantitative polymerase chain reaction (qPCR) with bone marrow specimens from AML patients. Then, Kaplan-Meier (KM) and Receiver Operating Characteristic (ROC) curves were used to substantiate the prognostic association between FOXN3-AS1 and AML patients within the TCGA database. Correlation between FOXN3-AS1 expression and gene mutation, immune, and immune function using Spearman correlation analysis. To explore the physical and functional interaction between FOXN3-AS1 and the DNMT1 protein, we utilized the RPISeq web tool from Iowa State University. Subsequently, we performed qPCR experiments to test the effect of 5AzaC (DNMT1 inhibitor) on FOXN3-AS1 expression AML cell lines (THP1 and OCI-AML3). We leveraged the "OncoPredict" R package in conjunction with the Genomics of Drug Sensitivity (GDSC) database to predict drug response in AML patients expressing FOXN3-AS1. We observed a significant upregulation of FOXN3-AS1 expression in AML patients compared to healthy controls using clinical samples. The TCGA database revealed an association between high FOXN3-AS1 expression and adverse prognosis. In our subsequent analysis, genes with poor prognostic implications in AML patients were exclusively identified in the FOXN3-AS1 high-expression group, further corroborating this relationship. AML patients with higher FOXN3-AS1 expression levels may respond less optimally to immunotherapy than patients with lower levels. Besides, we computationally predicted the interaction of FOXN3- AS1 and DNMT1 protein and experimentally confirmed that DNMT1i (GSK-3484862) affects the expression level of FOXN3-AS1. We also found that the chemotherapy drugs (5-Fluorouralic, Cisplatin, Dactolisib, Sapitinib, Temozolomide, Ulixertinib, Vinorelbine, Ruxolitinib, Osimertinib and Cisplatin) showed favorable responses in AML patients with high FOXN3-AS1 expression levels. Our candidate approach identifies FOXN3-AS1 as a prognostic indicator of survival in AML with a potential immune-related role. The preliminary observations we made on FOXN3-AS1/DNMT1 crosstalk warrant more in-depth invested immunotherapeutic approaches in AML.

RAG-seq: A NSR Primed and Transposase Tagmentation Mediated Strand-specific Total RNA Sequencing in Single Cell.

Single-cell RNA sequencing (scRNA-seq) has transformed our understanding of cellular diversity with unprecedented resolution. However, many current methods are limited in capturing full-length transcripts and discerning strand orientation. We present RAG-seq, an innovative strand-specific total RNA sequencing technique that combines not-so-random (NSR) primers with Tn5 transposase-mediated tagmentation. RAG-seq overcomes previous limitations by delivering comprehensive transcript coverage and maintaining strand orientation, which is essential for accurate quantification of overlapping genes and detection of antisense transcripts. Through optimized reverse transcription with oligo dT primers, rRNA depletion via Depletion of Abundant Sequences by Hybridization (DASH), and linear amplification, RAG-seq enhances sensitivity and reproducibility, especially for low-input samples and single cells. Application to mouse oocytes and early embryos highlights RAG-seq's superior performance in identifying stage-specific antisense transcripts, shedding light on their regulatory roles during early development. This advancement represents a significant leap in transcriptome analysis within complex biological contexts.

Pathological R-loops in bacteria from engineered expression of endogenous antisense RNAs whose synthesis is ordinarily terminated by Rho.

In many bacteria, the essential factors Rho and NusG mediate termination of synthesis of nascent transcripts (including antisense RNAs) that are not being simultaneously translated. It has been proposed that in Rho's absence toxic RNA-DNA hybrids (R-loops) may be generated from nascent untranslated transcripts, and genome-wide mapping studies in Escherichia coli have identified putative loci of R-loop formation from more than 100 endogenous antisense transcripts that are synthesized only in a Rho-deficient strain. Here we provide evidence that engineered expression in wild-type E. coli of several such individual antisense regions on a plasmid or the chromosome generates R-loops that, in an RNase H-modulated manner, serve to disrupt genome integrity. Rho inhibition was associated with increased prevalence of antisense R-loops also in Xanthomonas oryzae pv. oryzae and Caulobacter crescentus. Our results confirm the essential role of Rho in several bacterial genera for prevention of toxic R-loops from pervasive yet cryptic endogenous antisense transcripts. Engineered antisense R-looped regions may be useful for studies on both site-specific impediments to bacterial chromosomal replication and the mechanisms of their resolution.

Complex sporulation-specific expression of transcription termination factor Rho highlights its involvement in Bacillus subtilis cell differentiation

Termination factor Rho, responsible for the main factor-dependent pathway of transcription termination and the major inhibitor of antisense transcription, is an emerging regulator of various physiological processes in microorganisms. In Gram-positive bacterium Bacillus subtilis, Rho is involved in the control of cell adaptation to starvation and, in particular, in the control of sporulation, a complex differentiation program leading to formation of a highly resistant dormant spore. While the initiation of sporulation requires a decrease in Rho protein levels during the transition to stationary phase, the mechanisms regulating the expression of rho gene throughout the cell cycle remain largely unknown. Here we show that a drop in the activity of the vegetative SigA-dependent rho promoter causes the inhibition of rho expression in stationary phase. However, after the initiation of sporulation, rho gene is specifically reactivated in two compartments of the sporulating cell using distinct mechanisms. In the mother cell, rho expression occurs by read-through transcription initiated at the SigH-dependent promoter of the distal spo0F gene. In the forespore, rho gene is transcribed from the intrinsic promoter recognized by the alternative sigma factor SigF. These regulatory elements ensure the activity of Rho during sporulation, which appears important for the proper formation of spores. We provide experimental evidence that disruption of the spatiotemporal expression of rho during sporulation affects the resistance properties of spores, their morphology, and the ability to return to vegetative growth under favorable growth conditions.

Full-Length Transcriptome Profiling of the Complete Mitochondrial Genome of Sericothrips houjii (Thysanoptera: Thripidae: Sericothripinae) Featuring Extensive Gene Rearrangement and Duplicated Control Regions.

The mitochondrial genome (mitogenome) of Thysanoptera has extensive gene rearrangement, and some species have repeatable control regions. To investigate the characteristics of the gene expression, transcription and post-transcriptional processes in such extensively gene-rearranged mitogenomes, we sequenced the mitogenome and mitochondrial transcriptome of Sericothrips houjii to analyze. The mitogenome was 14,965 bp in length and included two CRs contains 140 bp repeats between COIII-trnN (CR1) and trnT-trnP (CR2). Unlike the putative ancestral arrangement of insects, S. houjii exhibited only six conserved gene blocks encompassing 14 genes (trnL2-COII, trnD-trnK, ND2-trnW, ATP8-ATP6, ND5-trnH-ND4-ND4L and trnV-lrRNA). A quantitative transcription map showed the gene with the highest relative expression in the mitogenome was ND4-ND4L. Based on analyses of polycistronic transcripts, non-coding RNAs (ncRNAs) and antisense transcripts, we proposed a transcriptional model of this mitogenome. Both CRs contained the transcription initiation sites (TISs) and transcription termination sites (TTSs) of both strands, and an additional TIS for the majority strand (J-strand) was found within antisense lrRNA. The post-transcriptional cleavage processes followed the "tRNA punctuation" model. After the cleavage of transfer RNAs (tRNAs), COI and ND3 matured as bicistronic mRNA COI/ND3 due to the translocation of intervening tRNAs, and the 3' untranslated region (UTR) remained in the mRNAs for COII, COIII, CYTB and ND5. Additionally, isoform RNAs of ND2, srRNA and lrRNA were identified. In summary, the relative mitochondrial gene expression levels, transcriptional model and post-transcriptional cleavage process of S. houjii are notably different from those insects with typical mitochondrial gene arrangements. In addition, the phylogenetic tree of Thripidae including S. houjii was reconstructed. Our study provides insights into the phylogenetic status of Sericothripinae and the transcriptional and post-transcriptional regulation processes of extensively gene-rearranged insect mitogenomes.

Antisense transcription from stress-responsive transcription factors fine-tunes the cold response in Arabidopsis.

Transcription of antisense long noncoding RNAs (lncRNAs) occurs pervasively across eukaryotic genomes. Only a few antisense lncRNAs have been characterized and shown to control biological processes, albeit with idiosyncratic regulatory mechanisms. Thus, we largely lack knowledge about the general role of antisense transcription in eukaryotic organisms. Here, we characterized genes with antisense transcription initiating close to the poly(A) signal of genes (PAS genes) in Arabidopsis (Arabidopsis thaliana). We compared plant native elongation transcript sequencing (plaNET-seq) with RNA sequencing during short-term cold exposure and detected massive differences between the response in active transcription and steady-state levels of PAS gene-derived mRNAs. The cold-induced expression of transcription factors B-BOX DOMAIN PROTEIN28 (BBX28) and C2H2-TYPE ZINC FINGER FAMILY PROTEIN5 (ZAT5) was detected by plaNET-seq, while their steady-state level was only slightly altered due to high mRNA turnover. Knockdown of BBX28 and ZAT5 or of their respective antisense transcripts severely compromised plant freezing tolerance. Decreased antisense transcript expression levels resulted in a reduced cold response of BBX28 and ZAT5, revealing a positive regulatory role of both antisense transcripts. This study expands the known repertoire of noncoding transcripts. It highlights that native transcription approaches can complement steady-state RNA techniques to identify biologically relevant players in stress responses.

Biological and clinical relevance of correlated expression levels of coding and long noncoding RNAs in HPV16 positive cervical cancers

Human papillomavirus (HPV) drives cervical cancer (CaCx) pathogenesis and viral oncoproteins jeopardize global gene expression in such cancers. In this study, our aim was to identify differentially expressed coding (DEcGs) and long noncoding RNA genes (DElncGs) specifically sense intronic and Natural Antisense Transcripts as they are located in the genic regions and may have a direct influence on the expression pattern of their neighbouring coding genes. We compared HPV16-positive CaCx patients (N = 44) with HPV-negative normal individuals (N = 34) by employing strand-specific RNA-seq and determined the relationships between DEcGs and DElncGs and their clinical implications. By performing Gene set enrichment and protein–protein interaction (PPI) analyses of DEcGs, we identified enrichment of processes crucial for abortive virus life cycle and cancer progression. The DEcGs formed 16 gene clusters which we identified through Molecular Complex Detection (MCODE) plugin of Cytoscape. All the gene clusters portrayed cancer-related functions. We recorded significantly correlated expression levels of 79 DElncGs with DEcGs at proximal genomic loci based on Pearson’s Correlation coefficients. Of these gene pairs, 24 pairs portrayed significantly altered correlation coefficients among patients, compared to normal individuals. Of these, 6 DEcGs of 6 such gene pairs, belonged to 5 of the identified gene clusters, one of which was survival-associated. Out of the 24 correlated DEcG: DElncG pairs, we identified 3 pairs, where expression of both members was significantly associated with patient overall survival. The findings justify the cooperative roles of these gene pairs, in patient prognostication, thereby bearing immense potential for translation. Thus, elucidation of correlative strengths between paired DElncGs and DEcGs in patient and normal samples, could serve as a foundation for identification of therapeutic and prognostic targets of HPV16-positive CaCx.Graphical abstract

Global Distribution and Diversity of Haloarchaeal pL6-Family Plasmids.

Australian isolates of Haloquadratum walsbyi, a square-shaped haloarchaeon, often harbor small cryptic plasmids of the pL6-family, approximately 6 kb in size, and five examples have been previously described. These plasmids exhibit a highly conserved gene arrangement and encode replicases similar to those of betapleolipoviruses. To assess their global distribution and recover more examples for analysis, fifteen additional plasmids were reconstructed from the metagenomes of seven hypersaline sites across four countries: Argentina, Australia, Puerto Rico, and Spain. Including the five previously described plasmids, the average plasmid size is 6002 bp, with an average G+C content of 52.5%. The tetramers GGCC and CTAG are either absent or significantly under-represented, except in the two plasmids with the highest %G+C. All plasmids share a similar arrangement of genes organized as outwardly facing replication and ATPase modules, but variations were observed in some core genes, such as F2, and some plasmids had acquired accessory genes. Two plasmids, pCOLO-c1 and pISLA-c6, shared 92.7% nt identity despite originating from Argentina and Spain, respectively. Numerous metagenomic CRISPR spacers matched sequences in the fifteen reconstructed plasmids, indicating frequent invasion of haloarchaea. Spacers could be assigned to haloarchaeal genera by mapping their associated direct repeats (DR), with half of these matching Haloquadratum. Finally, strand-specific metatranscriptome (RNA-seq) data could be used to demonstrate the active transcription of two pL6-family plasmids, including antisense transcripts.

Endogenous Bornavirus-like Elements in Bats: Evolutionary Insights from the Conserved Riboviral L-Gene in Microbats and Its Antisense Transcription in Myotis daubentonii.

Bats are ecologically diverse vertebrates characterized by their ability to host a wide range of viruses without apparent illness and the presence of numerous endogenous viral elements (EVEs). EVEs are well preserved, expressed, and may affect host biology and immunity, but their role in bat immune system evolution remains unclear. Among EVEs, endogenous bornavirus-like elements (EBLs) are bornavirus sequences integrated into animal genomes. Here, we identified a novel EBL in the microbat Myotis daubentonii, EBLL-Cultervirus.10-MyoDau (short name is CV.10-MyoDau) that shows protein-level conservation with the L-protein of a Cultervirus (Wuhan sharpbelly bornavirus). Surprisingly, we discovered a transcript on the antisense strand comprising three exons, which we named AMCR-MyoDau. The active transcription in Myotis daubentonii tissues of AMCR-MyoDau, confirmed by RNA-Seq analysis and RT-PCR, highlights its potential role during viral infections. Using comparative genomics comprising 63 bat genomes, we demonstrate nucleotide-level conservation of CV.10-MyoDau and AMCR-MyoDau across various bat species and its detection in 22 Yangochiropera and 12 Yinpterochiroptera species. To the best of our knowledge, this marks the first occurrence of a conserved EVE shared among diverse bat species, which is accompanied by a conserved antisense transcript. This highlights the need for future research to explore the role of EVEs in shaping the evolution of bat immunity.

Identification of the Promoter Antisense Transcript Enhancing the Transcription of the Equine Herpesvirus-1 Immediate-Early Gene.

Equine herpesvirus-1 (EHV-1) causes respiratory diseases, abortion, and encephalomyelitis in horses. The EHV-1 immediate-early (IE) protein, essential for viral replication, is transactivated by the binding of a multiprotein complex including the open reading frame 12 (ORF12) and some host factors to the IE promoter region. Promoter-associated non-coding RNAs (pancRNAs), which are transcribed from bidirectional promoters, regulate the transcription of neighboring genes in mammals and pathogens. In this study, we identified a novel pancRNA transcribed from across the areas of the 5'-untranslated region and a promoter of EHV-1 IE and named it IE pancRNA. IE pancRNA and mRNA were simultaneously expressed in EHV-1-infected RN33B-A68B2M cells. This pancRNA was also transcribed in RK13 and E. Derm cells, which are highly susceptible to EHV-1 infection. Furthermore, IE pancRNA upregulated IE gene expression in the presence of ORF12, and stable expression of IE pancRNA increased the number of EHV-1-infected RN33B-A68B2M cells. These results suggest that IE pancRNAs facilitate EHV-1 proliferation by promoting IE gene expression.

Integration of CTCF loops, methylome, and transcriptome in differentiating LUHMES as a model for imprinting dynamics of the 15q11-q13 locus in human neurons.

Human cell line models, including the neuronal precursor line LUHMES, are important for investigating developmental transcriptional dynamics within imprinted regions, particularly the 15q11-q13 Angelman (AS) and Prader-Willi (PWS) syndrome locus. AS results from loss of maternal UBE3A in neurons, where the paternal allele is silenced by a convergent antisense transcript UBE3A-ATS, a lncRNA that terminates at PWAR1 in non-neurons. qRT-PCR analysis confirmed the exclusive and progressive increase in UBE3A-ATS in differentiating LUHMES neurons, validating their use for studying UBE3A silencing. Genome-wide transcriptome analyses revealed changes to 11 834 genes during neuronal differentiation, including the upregulation of most genes within the 15q11-q13 locus. To identify dynamic changes in chromatin loops linked to transcriptional activity, we performed a HiChIP validated by 4C, which identified two neuron-specific CTCF loops between MAGEL2-SNRPN and PWAR1-UBE3A. To determine if allele-specific differentially methylated regions (DMR) may be associated with CTCF loop anchors, whole genome long-read nanopore sequencing was performed. We identified a paternally hypomethylated DMR near the SNRPN upstream loop anchor exclusive to neurons and a paternally hypermethylated DMR near the PWAR1 CTCF anchor exclusive to undifferentiated cells, consistent with increases in neuronal transcription. Additionally, DMRs near CTCF loop anchors were observed in both cell types, indicative of allele-specific differences in chromatin loops regulating imprinted transcription. These results provide an integrated view of the 15q11-q13 epigenetic landscape during LUHMES neuronal differentiation, underscoring the complex interplay of transcription, chromatin looping, and DNA methylation. They also provide insights for future therapeutic approaches for AS and PWS.

How antisense transcripts can evolve to encode novel proteins

Protein coding features can emerge de novo in non coding transcripts, resulting in emergence of new protein coding genes. Studies across many species show that a large fraction of evolutionarily novel non-coding RNAs have an antisense overlap with protein coding genes. The open reading frames (ORFs) in these antisense RNAs could also overlap with existing ORFs. In this study, we investigate how the evolution an ORF could be constrained by its overlap with an existing ORF in three different reading frames. Using a combination of mathematical modeling and genome/transcriptome data analysis in two different model organisms, we show that antisense overlap can increase the likelihood of ORF emergence and reduce the likelihood of ORF loss, especially in one of the three reading frames. In addition to rationalising the repeatedly reported prevalence of de novo emerged genes in antisense transcripts, our work also provides a generic modeling and an analytical framework that can be used to understand evolution of antisense genes.

Context-dependent antisense transcription from a neighboring gene interferes with the expression of mNeonGreen as a functional in vivo fluorescent reporter in the chloroplast of Chlamydomonas reinhardtii.

Advancing chloroplast genetic engineering in Chlamydomonas reinhardtii remains challenging, decades after its first successful transformation. This study introduces the development of a chloroplast-optimized mNeonGreen fluorescent reporter, enabling in vivo observation through a sixfold increase in fluorescence via context-aware construct engineering. Our research highlights the influence of transcriptional readthrough and antisense mRNA pairing on post-transcriptional regulation, pointing to novel strategies foroptimizing heterologous gene expression. We further demonstrate the applicability of these insights using an accessible experimentation system using glass-bead transformation and reestablishment of photosynthesis using psbH mutants, focusing on the mitigation of transcriptional readthrough effects. By characterizing heterologous expression using regulatory elements such as PrrnS, 5'atpA, and 3' rbcL in a sense-transcriptional context, we further documented up to twofold improvement in fluorescence levels. Our findings contribute new tools for molecular biology research in the chloroplast and evidence fundamental gene regulation processes that could enable the development of more effective chloroplast engineering strategies. This work not only paves the way for more efficient genetic engineering of chloroplasts but also deepens our understanding of the regulatory mechanisms at play.

Next-generation sequencing applied to non-syndromic tooth agenesis: A systematic literature review

BackgroundTo explore the genetic profile of Non-Syndromic Tooth Agenesis (NSTA) detected by Next-Generation Sequencing (NGS). MethodsA systematic review exploring PubMed, ScienceDirect, BMC, LILACS, Scopus, Embase and Web of Science was conducted. Studies investigating the genetic alterations involved in NSTA detected by NGS were selected. Data were extracted and risks of biases were evaluated. ResultsAfter screening 191 articles, 4 articles were included in this study. The overall qualities of the studies were considered high for 3 papers and low for the other one. Among 636 permanent missing teeth, second premolars and lateral incisors were the most affected ones. Whole exome sequencing and gene panel sequencing focusing either on exons plus exon-intron boundaries, or only exons were focused. Mutations on MSX1, PAX9, EDA, EDAR, AXIN2, WNT10A, LAMA3, DKK1, COL17A1, IRF6, LRP6, CHD7, CREBBP, EVC, LEF1, ROR2, TBX22, FGFR1 and TP63 were identified. Allelic frequencies of some variants were reported only in polish and Turkish populations and showing both slight and important variations. Moreover, Functional analysis was performed only for LRP6 variant in Turkish population. The published in-silico analyses of the proteins corresponding to the mutated LAMA3, MSX1 and WNT10 genes revealed a truncated protein for MSX1 and disulfide bridges destabilization for LAMA3 and WNT10A. ConclusionsSeveral genes were associated with NSTA and the candidacy of novel genes was suggested. Further research focusing on the exon-intron boundaries and the impact of mutations on the antisense transcripts should be carried out to better puzzle out their impact on the protein translation and structure.