Transcription Termination Sites Research Articles

Characterization and expression of the microtubule associated protein tau gene isoforms and their impact on clinical outcomes in glioma patients

Gliomas are the most common tumors found in the central nervous system. They are classified based on their histopathological malignancy. Low-grade gliomas (LGG) can turn into glioblastomas (GBM), the most aggressive form of brain cancer with the worst prognosis. Previous studies have shown that microtubule-associated protein tau (MAPT) is increased in GBM and that reducing it can improve survival in animal models. On the other hand, elevated MAPT expression has been linked to increased survival in LGG, indicating its potential as a survival biomarker. However, previous studies have primarily focused on the canonical protein-coding transcripts and gene-level expression of MAPT, disregarding the diversity of its many isoforms. Many diseases in almost every tissue have been associated with a dysregulation of alternative splicing, either through an altered ratio of the expressed isoforms or by expressing aberrant transcripts with different alternative transcription start and termination sites. This study investigates the association of specific MAPT transcripts with survival in LGG and GBM using publicly available genomic datasets and performing statistical analyses. The results of the study indicated coding and several non-coding isoforms associated with overall survival (OS), disease-specific survival (DSS), and progression-free interval (PFI). After including the relevant predictors as covariates, it was observed that among the protein-coding isoforms, MAPT-205 and MAPT-201 were significantly associated with clinical survival endpoints, but with opposite trends. These findings highlight the importance of studying the role of MAPT isoforms in glioma cellular phenotypes, aggressiveness, and patient survival.

Elucidating the Role of Transcriptomic Networks and DNA Methylation in Collagen Deposition of Dezhou Donkey Skin.

DNA methylation represents a predominant epigenetic modification with broad implications in various biological functions. Its role is particularly significant in the process of collagen deposition, a fundamental aspect of dermal development in donkeys. Despite its critical involvement, the mechanistic insights into how DNA methylation influences collagen deposition in donkey skin remain limited. In this study, we employed whole genome bisulfite sequencing (WGBS) and RNA sequencing (RNA-seq) to investigate the epigenetic landscape and gene expression profiles in the dorsal skin tissues of Dezhou donkeys across three developmental stages: embryonic (YD), juvenile (2-year-old, MD), and mature (8-year-old, OD). Our analysis identified numerous differentially methylated genes that play pivotal roles in skin collagen deposition and overall skin maturation, including but not limited to COL1A1, COL1A2, COL3A1, COL4A1, COL4A2, GLUL, SFRP2, FOSL1, SERPINE1, MMP1, MMP2, MMP9, and MMP13. Notably, we observed an inverse relationship between gene expression and DNA methylation proximal to transcription start sites (TSSs), whereas a direct correlation was detected in regions close to transcription termination sites (TTSs). Detailed bisulfite sequencing analyses of the COL1A1 promoter region revealed a low methylation status during the embryonic stage, correlating with elevated transcriptional activity and gene expression levels. Collectively, our findings elucidate key genetic markers associated with collagen deposition in the skin of Dezhou donkeys, underscoring the significant regulatory role of DNA methylation. This research work contributes to the foundational knowledge necessary for the genetic improvement and selective breeding of Dezhou donkeys, aiming to enhance skin quality attributes.

Transcription termination and readthrough in African swine fever virus.

African swine fever virus (ASFV) is a nucleocytoplasmic large DNA virus (NCLDV) that encodes its own host-like RNA polymerase (RNAP) and factors required to produce mature mRNA. The formation of accurate mRNA 3' ends by ASFV RNAP depends on transcription termination, likely enabled by a combination of sequence motifs and transcription factors, although these are poorly understood. The termination of any RNAP is rarely 100% efficient, and the transcriptional "readthrough" at terminators can generate long mRNAs which may interfere with the expression of downstream genes. ASFV transcriptome analyses reveal a landscape of heterogeneous mRNA 3' termini, likely a combination of bona fide termination sites and the result of mRNA degradation and processing. While short-read sequencing (SRS) like 3' RNA-seq indicates an accumulation of mRNA 3' ends at specific sites, it cannot inform about which promoters and transcription start sites (TSSs) directed their synthesis, i.e., information about the complete and unprocessed mRNAs at nucleotide resolution. Here, we report a rigorous analysis of full-length ASFV transcripts using long-read sequencing (LRS). We systematically compared transcription termination sites predicted from SRS 3' RNA-seq with 3' ends mapped by LRS during early and late infection. Using in-vitro transcription assays, we show that recombinant ASFV RNAP terminates transcription at polyT stretches in the non-template strand, similar to the archaeal RNAP or eukaryotic RNAPIII, unaided by secondary RNA structures or predicted viral termination factors. Our results cement this T-rich motif (U-rich in the RNA) as a universal transcription termination signal in ASFV. Many genes share the usage of the same terminators, while genes can also use a range of terminators to generate transcript isoforms varying enormously in length. A key factor in the latter phenomenon is the highly abundant terminator readthrough we observed, which is more prevalent during late compared with early infection. This indicates that ASFV mRNAs under the control of late gene promoters utilize different termination mechanisms and factors to early promoters and/or that cellular factors influence the viral transcriptome landscape differently during the late stages of infection.

The repertoire and structure of adhesion GPCR transcript variants assembled from publicly available deep-sequenced human samples.

Alternative splicing and multiple transcription start and termination sites can produce a diverse repertoire of mRNA transcript variants from a given gene. While the full picture of the human transcriptome is still incomplete, publicly available RNA datasets have enabled the assembly of transcripts. Using publicly available deep sequencing data from 927 human samples across 48 tissues, we quantified known and new transcript variants, provide an interactive, browser-based application Splice-O-Mat and demonstrate its relevance using adhesion G protein-coupled receptors (aGPCRs) as an example. On average, 24 different transcript variants were detected for each of the 33 human aGPCR genes, and several dominant transcript variants were not yet annotated. Variable transcription starts and complex exon-intron structures encode a flexible protein domain architecture of the N- and Ctermini and the seven-transmembrane helix domain (7TMD). Notably, we discovered the first GPCR (ADGRG7/GPR128) with eight transmembrane helices. Both the N- and C terminus of this aGPCR were intracellularly oriented, anchoring the N terminus in the plasma membrane. Moreover, the assessment of tissue-specific transcript variants, also for other gene classes, in our application may change the evaluation of disease-causing mutations, as their position in different transcript variants may explain tissue-specific phenotypes.

Genomic and epigenetic characterization of the arsenic-induced oncogenic microRNA-21

As one of the first identified oncogenic microRNAs, the precise details concerning the transcriptional regulation and function of microRNA-21 (miR-21) are still not completely established. The miR-21 gene is situated on chromosome 17q23.2, positioned at the 3′-UTR of the gene that encodes vacuole membrane protein-1 (VMP1). In this current study, we presented evidence indicating that miR-21 possesses its own gene promoter, which can be found in the intron 10 of the VMP1 gene. Chromatin immunoprecipitation followed by global DNA sequencing (ChIP-seq) revealed the presence of a broad H3K4me3 peak spanning the entire gene body of the primary miR-21 and the existence of super-enhancer clusters in the close proximity to both the miR-21 gene promoter and the transcription termination site in arsenic (As3+)-induced cancer stem-like cells (CSCs) and human induced pluripotent stem cells (hiPSCs). In non-transformed human bronchial epithelial cells (BEAS-2B), As3+ treatment enhanced Nrf2 binding to both the host gene VMP1 of miR-21 and the miR-21 gene. Knockout of Nrf2 inhibited both the basal and As3+-induced expressions of miR-21. Furthermore, the As3+-enhanced Nrf2 peaks in ChIP-seq fully overlap with these super-enhancers enriched with H3K4me1 and H3K27ac in the miR-21 gene, suggesting that Nrf2 may coordinate with other transcription factors through the super-enhancers to regulate the expression of miR-21 in cellular response to As3+. These findings demonstrate the unique genetic and epigenetic characteristics of miR-21 and may provide insights into understanding the novel mechanisms linking environmental As3+ exposure and human cancers.

Refining the transcriptional landscapes for distinct clades of virulent phages infecting Pseudomonas aeruginosa.

The introduction of high-throughput sequencing has resulted in a surge of available bacteriophage genomes, unveiling their tremendous genomic diversity. However, our current understanding of the complex transcriptional mechanisms that dictate their gene expression during infection is limited to a handful of model phages. Here, we applied ONT-cappable-seq to reveal the transcriptional architecture of six different clades of virulent phages infecting Pseudomonas aeruginosa. This long-read microbial transcriptomics approach is tailored to globally map transcription start and termination sites, transcription units, and putative RNA-based regulators on dense phage genomes. Specifically, the full-length transcriptomes of LUZ19, LUZ24, 14-1, YuA, PAK_P3, and giant phage phiKZ during early, middle, and late infection were collectively charted. Beyond pinpointing traditional promoter and terminator elements and transcription units, these transcriptional profiles provide insights in transcriptional attenuation and splicing events and allow straightforward validation of Group I intron activity. In addition, ONT-cappable-seq data can guide genome-wide discovery of novel regulatory element candidates, including noncoding RNAs and riboswitches. This work substantially expands the number of annotated phage-encoded transcriptional elements identified to date, shedding light on the intricate and diverse gene expression regulation mechanisms in Pseudomonas phages, which can ultimately be sourced as tools for biotechnological applications in phage and bacterial engineering.

Genome-wide adenine N6-methylation map reveals epigenomic regulation of lipid accumulation in Nannochloropsis

Epigenetic marks on histones and DNA, such as DNA methylation at N6-adenine (6mA), play crucial roles in gene expression and genome maintenance, but their deposition and function in microalgae remain largely uncharacterized. Here, we report a genome-wide 6mA map for the model industrial oleaginous microalga Nannochloropsis oceanica produced by single-molecule real-time sequencing. Found in 0.1% of adenines, 6mA sites are mostly enriched at the AGGYV motif, more abundant in transposons and 3′ untranslated regions, and associated with active transcription. Moreover, 6mA gradually increases in abundance along the direction of gene transcription and shows special positional enrichment near splicing donor and transcription termination sites. Highly expressed genes tend to show greater 6mA abundance in the gene body than do poorly expressed genes, indicating a positive interaction between 6mA and general transcription factors. Furthermore, knockout of the putative 6mA methylase NO08G00280 by genome editing leads to changes in methylation patterns that are correlated with changes in the expression of molybdenum cofactor, sulfate transporter, glycosyl transferase, and lipase genes that underlie reductions in biomass and oil productivity. By contrast, knockout of the candidate demethylase NO06G02500 results in increased 6mA levels and reduced growth. Unraveling the epigenomic players and their roles in biomass productivity and lipid metabolism lays a foundation for epigenetic engineering of industrial microalgae.

Internal transcription termination widely regulates differential expression of operon-organized genes including ribosomal protein and RNA polymerase genes in an archaeon.

Genes organized within operons in prokaryotes benefit from coordinated expression. However, within many operons, genes are expressed at different levels, and the mechanisms for this remain obscure. By integrating PacBio-seq, dRNA-seq, Term-seq and Illumina-seq data of a representative archaeon Methanococcus maripaludis, internal transcription termination sites (ioTTSs) were identified within 38% of operons. Higher transcript and protein abundances were found for genes upstream than downstream of ioTTSs. For representative operons, these differences were confirmed by northern blotting, qRT-PCRand western blotting, demonstrating that these ioTTS terminations were functional. Of special interest, mutation of ioTTSs in ribosomal protein (RP)-RNA polymerase (RNAP) operons not only elevated expression of the downstream RNAP genes but also decreased production of the assembled RNAP complex, slowed whole cell transcription and translation, and inhibited growth. Overexpression of the RNAP subunits with a shuttle vector generated the similar physiological effects. Therefore, ioTTS termination is a general and physiologically significant regulatory mechanism of the operon gene expression. Because the RP-RNAP operons are found to be widely distributed in archaeal species, this regulatory mechanism could be commonly employed in archaea.

DNA methylation and gene expression changes in mouse mammary tissue during successive lactations: part I – the impact of inflammation

ABSTRACT Mastitis is among the main reasons women cease breastfeeding, which leads to them supplementing breast milk with artificial formula. In farm animals, mastitis results in significant economic losses and the premature culling of some animals. Nevertheless, researchers do not know enough about the effect of inflammation on the mammary gland. This article discusses the changes to DNA methylation in mouse mammary tissue caused by lipopolysaccharide-induced inflammation (4 h post-injection of lipopolysaccharide). We analysed the expression of some genes related to mammary gland function, epigenetic regulation, and the immune response. The analysis focused on three comparisons: inflammation during the first lactation, inflammation during second lactation with no history of inflammation, and inflammation during second lactation with previous inflammation. We identified differentially methylated cytosines (DMCs), differentially methylated regions (DMRs), and some differentially expressed genes (DEGs) for each comparison. The three comparisons shared some DEGs; however, few DMCs and only one DMR were shared. These observations suggest that inflammation is one of several factors affecting epigenetic regulation during successive lactations. Furthermore, the comparison between animals in second lactation with and without inflammation, with no inflammation history during first lactation showed a different pattern compared to the other conditions in this experiment. This indicates that inflammation history plays an important role in determining epigenetic changes. The data presented in this study suggest that lactation rank and previous inflammation history are equally important when explaining mammary tissue gene expression and DNA methylation changes. Abbreviations: RRBS, reduced representation bisulfite sequencing; RT–qPCR, real-time quantitative polymerase chain reaction; MEC, mammary epithelial cells; TSS, transcription start site; TTS, transcription termination site; UTR, untranslated region; SINE, short interspersed nuclear element; LINE, long interspersed nuclear element; CGI, CpG island; DEG, differentially expressed gene; DMC, differentially methylated cytosine; DMR, differentially methylated region; GO term, gene ontology term; MF, molecular function; BP, biological process

Long-read direct RNA sequencing reveals epigenetic regulation of chimeric gene-transposon transcripts in Arabidopsis thaliana

Transposable elements (TEs) are accumulated in both intergenic and intragenic regions in plant genomes. Intragenic TEs often act as regulatory elements of associated genes and are also co-transcribed with genes, generating chimeric TE-gene transcripts. Despite the potential impact on mRNA regulation and gene function, the prevalence and transcriptional regulation of TE-gene transcripts are poorly understood. By long-read direct RNA sequencing and a dedicated bioinformatics pipeline, ParasiTE, we investigated the transcription and RNA processing of TE-gene transcripts in Arabidopsis thaliana. We identified a global production of TE-gene transcripts in thousands of A. thaliana gene loci, with TE sequences often being associated with alternative transcription start sites or transcription termination sites. The epigenetic state of intragenic TEs affects RNAPII elongation and usage of alternative poly(A) signals within TE sequences, regulating alternative TE-gene isoform production. Co-transcription and inclusion of TE-derived sequences into gene transcripts impact regulation of RNA stability and environmental responses of some loci. Our study provides insights into TE-gene interactions that contributes to mRNA regulation, transcriptome diversity, and environmental responses in plants.

Full-length transcriptome profiling of Aphidius gifuensis mitochondrial genome with gene rearrangement and control region duplication

Although mitochondrial gene rearrangement has been observed in many insect lineages, little is known about how it affects mitochondrial gene transcription. To address this question, we first constructed a quantitative transcription map for Aphidius gifuensis, a species of parasitoid wasp known to have a highly rearranged mitochondrial genome (mitogenome) and two potential control regions (CRs). Based on this transcription map, we assessed the models of the mitochondrial transcription and post-transcription cleavage. We found that the J and N strand of this mitogenome differ significantly in transcriptional regulation. On the J strand, we found two transcription initiation sites (TISs), five transcription termination sites (TTSs), and six polycistronic primary transcripts whereas only one TIS, one TTS and one polycistronic primary transcript can be found on the N strand. Most of the non-coding regions of both strands were transcribed into primary transcripts and cleaved after transcription. The proposed mode of transcription of A. gifuensis was similar to that of Drosophila, a model organism with no gene rearrangement. And two rearranged gene clusters (trnI-CR1-trnM-CR2-trnQ and trnW-trnY-trnC) seemed to have little effects on the mode of transcription. In addition, our results revealed the presence of TISs in CR1 and CR2, implying that both CRs maybe required for transcriptional regulation. Analysis of the post-transcriptional cleavage process showed that there were both “forward cleavage” and “reverse cleavage” models in A. gifuensis, and more than one way of cleavages were found in three regions. The incomplete transcripts suggested that the direction of mitochondrial RNA degradation was from 5′ to 3′ end and supported the view of polyadenylation-dependent RNA degradation. Our study provides insights into the transcriptional and post-transcriptional regulation processes of highly rearranged insect mitogenomes.

PacBio full-length transcriptome analysis provides new insights into transcription of chloroplast genomes

ABSTRACT Chloroplast and mitochondrial DNA (cpDNA and mtDNA) are apart from nuclear DNA (nuDNA) in a eukaryotic cell. The transcription system of chloroplasts differs from those of mitochondria and eukaryotes. In contrast to nuDNA and animal mtDNA, the transcription of cpDNA is still not well understood, primarily due to the unresolved identification of transcription initiation sites (TISs) and transcription termination sites (TTSs) on the genome scale. In the present study, we characterized the transcription of chloroplast (cp) genes with greater accuracy and comprehensive information using PacBio full-length transcriptome data from Arabidopsis thaliana. The major findings included the discovery of four types of artifacts, the validation and correction of cp gene annotations, the exact identification of TISs that start with G, and the discovery of polyA-like sites as TTSs. Notably, we proposed a new model to explain cp transcription initiation and termination at the whole-genome level. Four types of artifacts, degraded RNAs and splicing intermediates deserve the attention from researchers working with PacBio full-length transcriptome data, as these contaminant sequences can lead to incorrect downstream analysis. Cp transcription initiates at multiple promoters and terminates at polyA-like sites. Our study provides new insights into cp transcription and new clues to study the evolution of promoters, TISs, TTSs and polyA tails of eukaryotic genes.

DNA methylation and gene expression changes in mouse mammary tissue during successive lactations: part II – the impact of lactation rank

ABSTRACT Mastitis is among the main reasons women cease breastfeeding. In farm animals, mastitis results in significant economic losses and the premature culling of some animals. Nevertheless, the effect of inflammation on the mammary gland is not completely understood. This article discusses the changes to DNA methylation in mouse mammary tissue caused by lipopolysaccharide-induced inflammation after in vivo intramammary challenges and the differences in DNA methylation between 1st and 2nd lactations. Lactation rank induces 981 differential methylations of cytosines (DMCs) in mammary tissue. Inflammation in 1st lactation compared to inflammation in 2nd lactation results in the identification of 964 DMCs. When comparing inflammation in 1st vs. 2nd lactations with previous inflammation history, 2590 DMCs were identified. Moreover, Fluidigm PCR data show changes in the expression of several genes related to mammary function, epigenetic regulation, and the immune response. We show that the epigenetic regulation of two successive physiological lactations is not the same in terms of DNA methylation and that the effect of lactation rank on DNA methylation is stronger than that of the onset of inflammation. The conditions presented here show that few DMCs are shared between comparisons, suggesting a specific epigenetic response depending on lactation rank, the presence of inflammation, and even whether the cells had previously suffered inflammation. In the long term, this information could lead to a better understanding of the epigenetic regulation of lactation in both physiological and pathological conditions. Abbreviations: RRBS, reduced representation bisulphite sequencing; RT-qPCR, real-time quantitative polymerase chain reaction; MEC, mammary epithelial cells; MaSC, mammary stem cell; TSS, transcription start site; TTS, transcription termination site; UTR, untranslated region; SINE, short interspersed nuclear element; LINE, long interspersed nuclear element; CGI, CpG island; DEG, differentially expressed gene; DMC, differentially methylated cytosine; DMR, differentially methylated region; GO term, gene ontology term; MF, molecular function; BP, biological process

Splicing Characterization and Isoform Switch Events in Human Keratinocytes Carrying Oncogenes from High-Risk HPV-16 and Low-Risk HPV-84.

Infection of epithelial cells with high-risk HPV (HR-HPV) types, followed by expression of virus oncogenic proteins (E5, E6, and E7), leads to genomic imbalance, suppression of tumor inhibitors, and induction of oncogenes. Low-risk HPV (LR-HPV) may slow the rate at which cervical cancer spreads to an invasive stage since co-infection with LR-HPV is linked to a decreased risk of future invasive cancer than infection with HR-HPV alone. We then propose that cancer-progressing changes may be distinguished through identifying the functional differences between LR-HPV and HR-HPV. Lentiviral strategies were followed to establish HaCaT cells with constitutive expression of HPV oncogenes. RNAseq experiments were designed to analyze the transcriptome modulations caused by each of the E5, E6, and E7 oncogenes of HPV-16 and HPV-84 in HaCaT cells. We identified enhanced RNA degradation, spliceosome, and RNA polymerase pathways related to mRNA processing. ATTS (alternative transcription termination site) was discovered to be more prevalent in cells with HPV-16E5 than HPV-84E5. In HPV-16E6-infected cells, ATTS gain was significantly higher than ATTS loss. Cells with HPV-16E7 had more isoforms with intron retention (IR) than those with HPV-84E7. We identified switches in ADAM10, CLSPN, and RNPS1 that led to greater expression of the coding isoforms in HR-HPV. The results of this work highlight differences between LR-HPV and HR-HPV in mRNA processing. Moreover, crucial cervical cancer-related switch events were detected.

Co-transcriptional genome surveillance by HUSH is coupled to termination machinery

The HUSH complex recognizes and silences foreign DNA such as viruses, transposons, and transgenes without prior exposure to its targets. Here, we show that endogenous targets of the HUSH complex fall into two distinct classes based on the presence or absence of H3K9me3. These classes are further distinguished by their transposon content and differential response to the loss of HUSH. A de novo genomic rearrangement at the Sox2 locus induces a switch from H3K9me3-independent to H3K9me3-associated HUSH targeting, resulting in silencing. We further demonstrate that HUSH interacts with the termination factor WDR82 and-via its component MPP8-with nascent RNA. HUSH accumulates at sites of high RNAPII occupancy including long exons and transcription termination sites in a manner dependent on WDR82 and CPSF. Together, our results uncover the functional diversity of HUSH targets and show that this vertebrate-specific complex exploits evolutionarily ancient transcription termination machinery for co-transcriptional chromatin targeting and genome surveillance.

Multilevel interrogation of H3.3 reveals a primordial role in transcription regulation

BackgroundEukaryotic cells can rapidly adjust their transcriptional profile in response to molecular needs. Such dynamic regulation is, in part, achieved through epigenetic modifications and selective incorporation of histone variants into chromatin. H3.3 is the ancestral H3 variant with key roles in regulating chromatin states and transcription. Although H3.3 has been well studied in metazoans, information regarding the assembly of H3.3 onto chromatin and its possible role in transcription regulation remain poorly documented outside of Opisthokonts.ResultsWe used the nuclear dimorphic ciliate protozoan, Tetrahymena thermophila, to investigate the dynamics of H3 variant function in evolutionarily divergent eukaryotes. Functional proteomics and immunofluorescence analyses of H3.1 and H3.3 revealed a highly conserved role for Nrp1 and Asf1 histone chaperones in nuclear influx of histones. Cac2, a putative subunit of H3.1 deposition complex CAF1, is not required for growth, whereas the expression of the putative ortholog of the H3.3-specific chaperone Hir1 is essential in Tetrahymena. Our results indicate that Cac2 and Hir1 have distinct localization patterns during different stages of the Tetrahymena life cycle and suggest that Cac2 might be dispensable for chromatin assembly. ChIP-seq experiments in growing Tetrahymena show H3.3 enrichment over the promoters, gene bodies, and transcription termination sites of highly transcribed genes. H3.3 knockout followed by RNA-seq reveals large-scale transcriptional alterations in functionally important genes.ConclusionOur results provide an evolutionary perspective on H3.3’s conserved role in maintaining the transcriptional landscape of cells and on the emergence of specialized chromatin assembly pathways.

Premature termination of transcription is shaped by Rho and translated uORFS in Mycobacterium tuberculosis.

Little is known about the decisions behind transcription elongation versus termination in the human pathogen Mycobacterium tuberculosis (M.TB). By applying Term-seq to M.TB we found that the majority of transcription termination is premature and associated with translated regions, i.e., within previously annotated or newly identified open reading frames. Computational predictions and Term-seq analysis, upon depletion of termination factor Rho, suggests that Rho-dependent transcription termination dominates all transcription termination sites (TTS), including those associated with regulatory 5' leaders. Moreover, our results suggest that tightly coupled translation, in the form of overlapping stop and start codons, may suppress Rho-dependent termination. This study provides detailed insights into novel M.TB cis-regulatory elements, where Rho-dependent, conditional termination of transcription and translational coupling together play major roles in gene expression control. Our findings contribute to a deeper understanding of the fundamental regulatory mechanisms that enable M.TB adaptation to the host environment offering novel potential points of intervention.

Analysis of Fowl Adenovirus 4 Transcriptome by De Novo ORF Prediction Based on Corrected Nanopore Full-Length cDNA Sequencing Data.

The transcriptome of fowl adenovirus has not been comprehensively revealed. Here, we attempted to analyze the fowl adenovirus 4 (FAdV-4) transcriptome by deep sequencing. RNA samples were extracted from chicken LMH cells at 12, 18 or 26 h post-FAdV-4 infection, and subjected to Illumina strand-specific RNA-seq or nanopore full-length PCR-cDNA sequencing. After removing the reads of host cells, the data of FAdV-4 nanopore full-length cDNAs (transcripts) were corrected with reads from the Illumina RNA-seq, mapped to the viral genome and then used to predict viral open reading frames (ORFs). Other than 42 known ORFs, 39 novel ORFs were annotated to the FAdV-4 genome. Different from human adenovirus 5, one FAdV-4 ORF was often encoded by several transcripts, and more FAdV-4 ORFs were located on two exons. With these data, 18 major transcription start sites and 15 major transcription termination sites were defined, implying 18 viral promoters and 15 polyadenylation signals. The temporal cascade of viral gene transcription was observed in FAdV-4-infected cells, with six promoters possessing considerable activity in the early phase. Unexpectedly, four promoters, instead of one major late promoter, were engaged in the transcription of the viral genus-common genes on the forward strand. The clarification of the FAdV-4 transcriptome laid a solid foundation for the study of viral gene function, virulence and virus evolution, and it would help construct FAdV-4 as a gene transfer vehicle. The strategy of de novo ORF prediction could be used to parse the transcriptome of other novel adenoviruses.

Deciphering the influence of genome architecture in minimized bacteria

Stochastic gene expression begins with the repeated transcription of DNA by RNA polymerase (RNAP) to create the various functional forms (rRNA, tRNA, mRNA, and sRNA) of the bacterial transcriptome. The initiation and termination of transcriptional events are associated with the genome architecture, the local arrangement of genetic features along the circular bacterial genome identified via sequence motifs (promoters, Shine-Dalgarno, gene open-reading frames, and transcription termination sites, etc.). The local arrangement of these sequence motifs form operon-like regions, known as transcriptional units, directly controlling the expression of encoded RNA species. The stochastic activity between RNAP and the genetic features of a transcription unit results in differential transcription forming RNA isoforms, various RNA transcripts from a similar region of different lengths. Locations of the genetic features that define the transcription unit boundaries were predicted using structural and bioinformatic analysis, and then integrated to predict transcriptional units within two minimized bacteria, JCVI-syn1.0 and JCVI-syn3A. The theoretical predictions agree well with observations of expression obtained using Oxford Nanopore Technologies Direct RNA sequencing for Syn1.0 the precursor of Syn3A. Comparisons of the computational and experimental results help to provide new insight into transcriptional activity within the organisms, resolve the distribution of RNA isoform complexity evolved from key genomic regions such as those involved in membrane transport and cell division, motivate the investigation into the post-transcriptional activity of RNA, and explain the impact on gene expression from the genome reduction of Syn1.0 to Syn3A.

Unfolding the Bacterial Transcriptome Landscape Using Oxford Nanopore Technology Direct RNA Sequencing.

Current genome annotation ignores important features of the transcriptome, such as untranslated regions and operon maps. RNA sequencing (RNA-seq) helps in identifying such features; however, the fragmentation step of classical RNA-seq makes this task challenging. Long-read sequencing methods, such as that of Oxford Nanopore Technologies (ONT), enable the sequencing of intact RNA molecules. Here, we present a method to annotate the full features of bacterial transcriptomes by combining a modified ONT direct RNA-seq method with our computational pipeline, UNAGI bacteria. The method reveals the full complexity of the bacterial transcriptome landscape, including transcription start sites, transcription termination sites, operon maps, and novel genes.