Transcription End Sites Research Articles

3D chromatin structures associated with ncRNA roX2 for hyperactivation and coactivation across the entire X chromosome.

The three-dimensional (3D) organization of chromatin within the nucleus is crucial for gene regulation. However, the 3D architectural features that coordinate the activation of an entire chromosome remain largely unknown. We introduce an omics method, RNA-associated chromatin DNA-DNA interactions, that integrates RNA polymerase II (RNAPII)-mediated regulome with stochastic optical reconstruction microscopy to investigate the landscape of noncoding RNA roX2-associated chromatin topology for gene equalization to achieve dosage compensation. Our findings reveal that roX2 anchors to the target gene transcription end sites (TESs) and spreads in a distinctive boot-shaped configuration, promoting a more open chromatin state for hyperactivation. Furthermore, roX2 arches TES to transcription start sites to enhance transcriptional loops, potentially facilitating RNAPII convoying and connecting proximal promoter-promoter transcriptional hubs for synergistic gene regulation. These TESs cluster as roX2 compartments, surrounded by inactive domains for coactivation of multiple genes within the roX2 territory. In addition, roX2 structures gradually form and scaffold for stepwise coactivation in dosage compensation.

MYC Binding Near Transcriptional End Sites Regulates Basal Gene Expression, Read-Through Transcription and Intragenic Contacts.

The MYC oncoprotein regulates numerous genes involved in cellular processes such as cell cycle and mitochondrial and ribosomal structure and function. This requires heterodimerization with its partner, MAX, and binding to specific promoter and enhancer elements. Here, we show that MYC and MAX also bind near transcriptional end sites (TESs) of over one-sixth of all annotated genes. These interactions are dose-dependent, evolutionarily conserved, stabilize the normally short-lived MYC protein and regulate expression both in concert with and independent of MYC's binding elsewhere. MYC's TES binding occurs in association with other transcription factors, alters the chromatin landscape, increases nuclease susceptibility and can alter transcriptional read-through, particularly in response to certain stresses. MYC-bound TESs can directly contact promoters and may fine-tune gene expression in response to both physiologic and pathologic stimuli. Collectively, these findings support a previously unrecognized role for MYC in regulating transcription and its read-through via direct intragenic contacts between TESs and promoters.

Abstract 403: APOBEC3 enzymes induce chromosomal translocations in solid cancer

Abstract Introduction: Chromosomal translocations are common oncogenic drivers in human cancers, and an increasing number of translocations are being considered as crucial diagnostic and prognostic markers in clinics. In B-cell lymphoma, a number of chromosomal translocations are directed by the off-target activity of the activation-induced cytidine deaminase (AID; a B-cell specific apolipoprotein B mRNA-editing enzyme, catalytic polypeptode-like (APOBEC) family enzyme). However, in solid tumors, no such enzymes capable of directing chromosomal translocations have been identified. Given recent findings on APOBEC-mediated mutagenesis in solid cancers, APOBECs may be involved in the formation of chromosomal translocations in solid tumors, like AID does in B cells. In human, the APOBEC3 (A3) family consists of seven members (A3A-A3H) and is associated with C <t or="" c<g="" substitutions.="" recent="" evidence="" shows="" that="" a3a="" might="" be="" involved="" in="" chromosomal="" instability="" leading="" to="" drug="" resistance="" lung="" cancer.="" however,="" it="" has="" not="" been="" explored="" whether="" a3s="" generate="" dna="" dsbs,="" eventually="" translocations.="" this="" study,="" we="" investigated="" the="" role="" of="" and="" their="" mechanisms="" translocations="" solid="" cancer.Methods: We applied high-throughput genome-wide translocation sequencing (HTGTS) approach to map genome-wide chromosomal translocations induced by human A3s in mouse fibroblast and human epithelial cells. We correlated patterns of translocations induced by A3s with several other cellular features including transcription, DNA accessibility, DNA replication timing, and mutational signatures. We validated the contribution of A3 to the generation of oncogenic translocations in lung cancer. Results: Ectopic expression of A3s significantly increased chromosomal translocations in mouse fibroblasts and human epithelial cells. A3s and AID preferentially induced translocations in different parts of the genome, but some of the translocation hotspots were shared among them. Hierarchical clustering of hotspots showed that A3-mediated translocations were distinguished from AID-mediated translocations. Notably, the junction distribution of breakpoint clusters showed three distinct translocation patterns based on the locations of breakpoint clusters: (1) AID-preferred; (2) A3-preferred; and (3) AID/A3-mixed. AID-mediated translocations were heavily enriched near the transcription start site, whereas A3-mediated translocations were dispersed across the gene body and transcription end site. Furthermore, DNA replication timing and genomic architectures were linked to the formation of A3-mediated chromosomal translocations in solid cancer. Finally, A3 contributed to the formation of oncogenic rearrangements in lung cancer. Conclusions: A3 enzymes induced chromosomal translocations with distinct patterns compared to AID and facilitated chromosomal translocation formation in solid cancers. Citation Format: Taek-Chin Cheong, Qi Wang, Ahram Jang, Elif Karaca-Atabay, Roberto Chiarle. APOBEC3 enzymes induce chromosomal translocations in solid cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 403.

Abstract 404: LINE-1 retrotransposition promotes chromosomal translocations through RNA-templated DNA repair in human cancers

Abstract Introduction: Approximately 50% of human cancers have somatic integrations of long interspersed element-1 (LINE-1; also known as L1). L1 is a retrotransposon that copies itself through RNA and integrates into new genomic loci. Although L1 sequences comprise approximately 17% of the human genome, only 100-150 L1 loci have retained the ability to retrotranspose. These full-length, retrotransposition-competent L1s are typically repressed in human somatic cells, but there is evidence of their significant reactivation in human cancers. Therefore, L1 overexpression is a hallmark of many human cancers, particularly in highly malignant tumor types such as breast, ovarian, pancreatic, esophageal, lung, head-and-neck, colon, prostate, and liver cancers. Results: In many cancers, chromosomal translocations are fundamental pathogenetic events initiated by the generation of DNA double-strand breaks (DSBs). Although LINE-1 (L1) retrotransposition events have been implicated in chromosomal rearrangements associated with most human cancers, the frequency and mechanisms by which L1 is involved in these rearrangements, including chromosomal translocations, remain poorly characterized. By combining high-throughput genome-wide translocation sequencing (HTGTS) and two sequencing techniques to detect L1 insertion sites (PolyA-seq and CELTICS-seq), we demonstrated that the presence of an active human L1 retrotransposition significantly increases the frequency of chromosomal translocations. By identifying L1-dependent translocation hotspots in HEK293T cells we highlight the requirement for the L1 reverse transcriptase (RT) activity and the crucial role of pre-existing DNA DSBs in promoting L1-mediated chromosomal translocations. L1-dependent translocation hotspots showed a marked preference for transcription end sites (TES) of active genes, early replication timing regions, and accessible chromatin regions. Therefore, while cancer genome analysis provides a static view of chromosomal rearrangements based on canonical L1 integrations, our functional studies reveal an unanticipated predominance of chromosomal translocations facilitated by non-canonical L1 retrotransposition via RNA-templated repair of existing DSBs, largely expanding our knowledge on the mechanism by which L1 retrotransposition induces genomic instability in human cancers. Citation Format: Jianli Tao, Luca Alessandri, Carlos Mendez-Dorantes, Kathleen H. Burns, Qi Wang, Roberto Chiarle. LINE-1 retrotransposition promotes chromosomal translocations through RNA-templated DNA repair in human cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 404.

Global repair is the primary nucleotide excision repair subpathway for the removal of pyrimidine-pyrimidone (6-4) damage from the Arabidopsis genome

Ultraviolet (UV) component of solar radiation impairs genome stability by inducing the formation of pyrimidine-pyrimidone (6-4) photoproducts [(6-4)PPs] in plant genomes. (6-4)PPs disrupt growth and development by interfering with transcription and DNA replication. To resist UV stress, plants employ both photoreactivation and nucleotide excision repair that excises oligonucleotide containing (6-4)PPs through two subpathways: global and transcription-coupled excision repair (TCR). Here, we analyzed the genome-wide excision repair-mediated repair of (6-4)PPs in Arabidopsis thaliana and found that (6-4)PPs can be repaired by TCR; however, the main subpathway to remove (6-4)PPs from the genome is global repair. Our analysis showed that open chromatin genome regions are more rapidly repaired than heterochromatin regions, and the repair level peaks at the promoter, transcription start site and transcription end site of genes. Our study revealed that the repair of (6-4)PP in plants showed a distinct genome-wide repair profile compared to the repair of other major UV-induced DNA lesion called cyclobutane pyrimidine dimers (CPDs).

The plant POLYMERASE-ASSOCIATED FACTOR1 complex links transcription and H2B monoubiquitination genome wide.

The evolutionarily conserved POLYMERASE-ASSOCIATED FACTOR1 complex (Paf1C) participates in transcription, and research in animals and fungi suggests that it facilitates RNA POLYMERASE II (RNAPII) progression through chromatin. We examined the genomic distribution of the EARLY FLOWERING7 (ELF7) and VERNALIZATION INDEPENDENCE3 subunits of Paf1C in Arabidopsis (Arabidopsis thaliana). The occupancy of both subunits was confined to thousands of gene bodies and positively associated with RNAPII occupancy and the level of gene expression, supporting a role as a transcription elongation factor. We found that monoubiquitinated histone H2B, which marks most transcribed genes, was strongly reduced genome wide in elf7 seedlings. Genome-wide profiling of RNAPII revealed that in elf7 mutants, RNAPII occupancy was reduced throughout the gene body and at the transcription end site of Paf1C-targeted genes, suggesting a direct role for the complex in transcription elongation. Overall, our observations suggest a direct functional link between Paf1C activity, monoubiquitination of histone H2B, and the transition of RNAPII to productive elongation. However, for several genes, Paf1C may also act independently of H2Bub deposition or occupy these genes more stable than H2Bub marking, possibly reflecting the dynamic nature of Paf1C association and H2Bub turnover during transcription.

Functional Dissection of Cellular Programs to Uncover Novel Gene Dependencies in AML

Genome-scale CRISPR-Cas9 screens have the power to unveil the Achilles' heel of neoplastic cells. Typically, analyses of such large-scale data sets focus on single gene dependencies. An alternative strategy is to evaluate functional networks enriched in a disease or molecular subset of interest. We applied this strategy to the Broad Institute's Cancer Dependency Map (DepMap) data set consisting of genome-scale CRISPR-Cas9 screens in over 1000 cancer cell line models. We hypothesized that interrogating enriched pathways with context-specific dependencies leads to the discovery of functionally informed gene dependencies. Single sample gene set enrichment analysis (ssGSEA) of over 1000 cancer cell lines in the DepMap identified hematologic malignancies to be highly enriched for signatures associated with elevated transcriptional activity. Notably, within hematologic malignancies, acute myeloid leukemia (AML) with a rearrangement in the KMT2A gene ( KMT2Ar-AML) was the most significantly enriched for dependency on transcriptional activity-associated gene signatures. We next interrogated KMT2Ar-AML gene dependencies (excluding common essential genes) with the human kinase database KinMap (http://www.kinhub.org/kinmap/) to identify readily druggable genes involved in transcriptional activity. Cyclin-dependent kinase 13 ( CDK13) was the only gene meeting these criteria. Further DepMap data analysis confirmed that KMT2Ar-AML cell lines were indeed more strongly dependent on CDK13 than all other cancer cell lines screened. Moreover, when we performed GSEA on the gene dependencies observed in CDK13-dependent versus non-dependent cell lines, we found transcriptional activity and KMT2A-target gene sets as top hits. We next validated AML cell dependency on CDK13 with orthogonal genetic approaches (CRISPR-Cas9 knock-out and shRNA) in vitro in human AML cell lines and cells from patient-derived xenograft (PDX) models of KMT2Ar-AML. Perturbation of CDK13 induced cell death with hallmarks of apoptosis. Doxycycline-inducible shRNA directed against CDK13 also impaired AML progression in the peripheral blood and bone marrow in an MV4-11- KMT2Ar xenograft. Moreover, in an orthotopic PDX model, doxycycline inducible knock-out of CDK13 in mice with established disease significantly reduced leukemia burden in the peripheral blood and bone marrow. CDK13 is reported to regulate transcriptional elongation and the clearance of prematurely terminated RNAs. To decipher why AML cells, particularly KMT2Ar, need CDK13 to survive, we identified the CDK13 binding sites and histone marks involved in transcriptional regulation in AML using CUT and RUN and ChIP-seq: We determined that CDK13 co-localizes with H3K4me3 and H3K27ac at promoter sites. Global gene expression studies following the knock-out of CDK13 revealed that the majority of CDK13 bound genes were also differentially expressed, with significantly more genes decreased than increased in expression after CDK13 knock-out. GSEA of CDK13-bound genes with decreased expression upon CDK13 knock-out identified gene signatures associated with transcriptional activation and KMT2A-targets. We next studied the characteristics of CDK13 target genes and observed a predominance of long genes (total length of gene exons > 10 kb, GRCh38.p12). GSEA of genes ranked by size, confirmed that longer genes were enriched for CDK13 target genes, CDK13 differential AML dependencies, and for KMT2A-target transcriptional signatures. Additionally, following CDK13 perturbation in KMT2Ar-AML we observed a decrease of Pol2/pPol2Ser2 occupancy at the transcription end site of long genes. In summary, using functional pathway analyses, we identified CDK13 as a candidate gene dependency enriched in KMT2Ar-AML, a target not immediately prioritized with single gene analysis. Mechanistically, our data support that CDK13 controls Pol2 processivity, with CDK13 perturbation leading to a decreased occupancy at the 3' end of long genes. KMT2Ar, CDK13 dependent cells are also enriched for dependency on long genes, thereby suggesting a molecular basis for the CDK13 dependency in KMT2Ar-AML.

CCIVR2 facilitates comprehensive identification of both overlapping and non-overlapping antisense transcripts within specified regions

Pairs of sense and antisense transcriptions that are adjacent at their 5′ and 3′ regions are called divergent and convergent transcription, respectively. However, the structural properties of divergent/convergent transcription in different species or RNA biotypes are poorly characterized. Here, we developed CCIVR2, a program that facilitates identification of both overlapping and non-overlapping antisense transcripts produced from divergent/convergent transcription whose transcription start sites (TSS) or transcript end sites (TES) are located within a specified region. We used CCIVR2 to analyze antisense transcripts starting around the sense TSS (from divergent transcription) or ending around the sense TES (from convergent transcription) in 11 different species and found species- and RNA biotype-specific features of divergent/convergent transcription. Furthermore, we confirmed that CCIVR2 enables the identification of multiple sense/antisense transcript pairs from divergent transcription, including those with known functions in processes such as embryonic stem cell differentiation and TGFβ stimulation. CCIVR2 is therefore a valuable bioinformatics tool that facilitates the characterization of divergent/convergent transcription in different species and aids the identification of functional sense/antisense transcript pairs from divergent transcription in specified biological processes.

Accessible gene borders establish a core structural unit for chromatin architecture in Arabidopsis.

Three-dimensional (3D) chromatin structure is linked to transcriptional regulation in multicellular eukaryotes including plants. Taking advantage of high-resolution Hi-C (high-throughput chromatin conformation capture), we detected a small structural unit with 3D chromatin architecture in the Arabidopsis genome, which lacks topologically associating domains, and also in the genomes of tomato, maize, and Marchantia polymorpha. The 3D folding domain unit was usually established around an individual gene and was dependent on chromatin accessibility at the transcription start site (TSS) and transcription end site (TES). We also observed larger contact domains containing two or more neighboring genes, which were dependent on accessible border regions. Binding of transcription factors to accessible TSS/TES regions formed these gene domains. We successfully simulated these Hi-C contact maps via computational modeling using chromatin accessibility as input. Our results demonstrate that gene domains establish basic 3D chromatin architecture units that likely contribute to higher-order 3D genome folding in plants.

Deep annotation of long noncoding RNAs by assembling RNA-seq and small RNA-seq data

Long noncoding RNAs (lncRNAs) are increasingly being recognized as modulators in various biological processes. However, due to their low expression, their systematic characterization is difficult to determine. Here, we performed transcript annotation by a newly developed computational pipeline, termed RNA-seq and small RNA-seq combined strategy (RSCS), in a wide variety of cellular contexts. Thousands of high-confidence potential novel transcripts were identified by the RSCS, and the reliability of the transcriptome was verified by analysis of transcript structure, base composition, and sequence complexity. Evidenced by the length comparison, the frequency of the core promoter and the polyadenylation signal motifs, and the locations of transcription start and end sites, the transcripts appear to be full length. Furthermore, taking advantage of our strategy, we identified a large number of endogenous retrovirus-associated lncRNAs, and a novel endogenous retrovirus-lncRNA that was functionally involved in control of Yap1 expression and essential for early embryogenesis was identified. In summary, the RSCS can generate a more complete and precise transcriptome, and our findings greatly expanded the transcriptome annotation for the mammalian community.

Hybrid sequencing discloses unique aspects of the transcriptomic architecture in equid alphaherpesvirus 1

This study employed both short-read sequencing (SRS, Illumina) and long-read sequencing (LRS Oxford Nanopore Technologies) platforms to conduct a comprehensive analysis of the equid alphaherpesvirus 1 (EHV-1) transcriptome. The study involved the annotation of canonical mRNAs and their transcript variants, encompassing transcription start site (TSS) and transcription end site (TES) isoforms, in addition to alternative splicing forms. Furthermore, the study revealed the presence of numerous non-coding RNA (ncRNA) molecules, including intergenic and antisense transcripts, produced by EHV-1. An intriguing finding was the abundant production of chimeric transcripts, some of which potentially encode fusion polypeptides. Moreover, EHV-1 exhibited a greater incidence of transcriptional overlaps and splicing compared to related viruses. It is noteworthy that many genes have their unique TESs along with the co-terminal transcription ends, a characteristic scarcely seen in other alphaherpesviruses. The study also identified transcripts that overlap the replication origins of the virus. Moreover, a novel ncRNA, referred to as NOIR, was found to intersect with the 5′-ends of longer transcript isoform specified by the major transactivator genes ORF64 and ORF65, surrounding the OriL. These findings together imply the existence of a key regulatory mechanism that governs both transcription and replication through, among others, a process that involves interference between the DNA and RNA synthesis machineries.

Interplay between PML NBs and HIRA for H3.3 dynamics following type I interferon stimulus.

Promyelocytic leukemia Nuclear Bodies (PML NBs) are nuclear membrane-less organelles physically associated with chromatin underscoring their crucial role in genome function. The H3.3 histone chaperone complex HIRA accumulates in PML NBs upon senescence, viral infection or IFN-I treatment in primary cells. Yet, the molecular mechanisms of this partitioning and its function in regulating histone dynamics have remained elusive. By using specific approaches, we identify intermolecular SUMO-SIM interactions as an essential mechanism for HIRA recruitment in PML NBs. Hence, we describe a role of PML NBs as nuclear depot centers to regulate HIRA distribution in the nucleus, dependent both on SP100 and DAXX/H3.3 levels. Upon IFN-I stimulation, PML is required for interferon-stimulated genes (ISGs) transcription and PML NBs become juxtaposed to ISGs loci at late time points of IFN-I treatment. HIRA and PML are necessary for the prolonged H3.3 deposition at the transcriptional end sites of ISGs, well beyond the peak of transcription. Though, HIRA accumulation in PML NBs is dispensable for H3.3 deposition on ISGs. We thus uncover a dual function for PML/PML NBs, as buffering centers modulating the nuclear distribution of HIRA, and as chromosomal hubs regulating ISGs transcription and thus HIRA-mediated H3.3 deposition at ISGs upon inflammatory response.

Genome-Wide Mapping of 5' Isoforms with 5'-Seq.

The transcriptome is far more complex than previously assumed. Transcripts from the same gene can differ in terms of transcription start site, transcription end site, or pattern of splicing, and growing evidence supports the functional importance of these distinct transcript isoforms. Easily identifying these isoforms experimentally via library construction and high-throughput sequencing is crucial. Current library construction methods for identifying transcription start sites (5' transcript isoforms) involve large number of steps and (expensive) reagents, utilization of cDNA intermediates for adapter ligation, and are less suitable for studying low-abundance isoforms. Here, I describe a quick protocol for the generation of sequencing libraries to define capped 5' isoforms (5'-Seq) of various abundances in yeast and suggest a 5' isoform data analysis pipeline. The protocol relies on the utilization of a dephosphorylation-decapping method (oligo-capping) to generate a sequencing library from mRNA fragments and is a simplification of previously published 5' isoform protocols in terms of the handling steps, time, and cost. This method is exemplified using Saccharomyces cerevisiae mRNA, but it can be applied to various cellular conditions to study the effects of 5' transcript isoforms on transcriptional and/or translational regulation. © 2023 Wiley Periodicals LLC. Basic Protocol: Construction of a DNA sequencing library from capped 5' isoforms Support Protocol: Sequencing data analysis.

MED26-containing Mediator may orchestrate multiple transcription processes through organization of nuclear bodies.

Mediator is a coregulatory complex that plays essential roles in multiple processes of transcription regulation. One of the human Mediator subunits, MED26, has a role in recruitment of the super elongation complex (SEC) to polyadenylated genes and little elongation complex (LEC) to non-polyadenylated genes, including small nuclear RNAs (snRNAs) and replication-dependent histone (RDH) genes. MED26-containing Mediator plays a role in 3' Pol II pausing at the proximal region of transcript end sites in RDH genes through recruitment of Cajal bodies (CBs) to histone locus bodies (HLBs). This finding suggests that Mediator is involved in the association of CBs with HLBs to facilitate 3' Pol II pausing and subsequent 3'-end processing by supplying 3'-end processing factors from CBs. Thus, we argue the possibility that Mediator is involved in the organization of nuclear bodies to orchestrate multiple processes of gene transcription.

Whole-genome DNA methylome analysis of different developmental stages of the entomopathogenic fungus Beauveria bassiana NCHU-157 by nanopore sequencing.

The entomopathogenic fungus (EPF), Beauveria bassiana, is an important and commonly used EPF for microbial control. However, the role of DNA methylation has not been thoroughly studied. Therefore, the whole genomic DNA methylome of one promising EPF isolate, B. bassiana NCHU-157 (Bb-NCHU-157), was investigated by Oxford Nanopore Technologies (ONT). First, the whole genome of Bb-NCHU-157 was sequenced by next-generation sequencing (NGS) and ONT. The genome of Bb-NCHU-157 contains 16 contigs with 34.19Mb and 50% GC content, which are composed of 10,848 putative protein-coding genes. Two putative DNA methyltransferases (DNMTs) were found, including Dim-2 and C-5 cytosine-specific DNA methylases. Both DNMTs showed higher expression levels in the mycelium stage than in the conidia stage, indicating that development of DNA methylation in Bb-NCHU-157 might occur in the mycelium stage. The global methylation level of the mycelium stage (5mC = 4.56%, CG = 3.33%, CHG = 0.74%, CHH = 0.49%) was higher than that of the conidial stage (5mC = 2.99%, CG = 1.99%, CHG = 0.63%, CHH = 0.37%) in both the gene and transposable element (TE) regions. Furthermore, the TE regions showed higher methylation frequencies than the gene regions, especially for CHH site methylation, suggesting regulation of genomic stabilization during mycelium development. In the gene regions, high methylation frequencies were found around the transcription start site (TSS) and transcription end site (TES). Moreover, CG and CHG methylation mainly occur in the promoter and intergenic regions, while CHH methylation occurs in the TE region. Among the methylated regions, 371, 661, and 756 differentially DNA methylated regions (DMRs) were hypermethylated in the mycelium in CG, CHG, and CHH, while only 13 and 7 DMRs were hypomethylated in the mycelium in CHG, and CHH, respectively. Genes located in the DMR shared the GO terms, DNA binding (GO: 0003677), and sequence-specific DNA binding (GO: 0043565) for hypermethylation in the mycelium, suggesting that methylation might regulate gene expression from the initial process. Evaluation of the DNA methylome in Bb-NCHU-157 by ONT provided new insight into this field. These data will be further validated, and epigenetic regulation during the development of B. bassiana will be explored.

New insights into genome annotation in Podospora anserina through re-exploiting multiple RNA-seq data

BackgroundPublicly available RNA-seq datasets are often underused although being helpful to improve functional annotation of eukaryotic genomes. This is especially true for filamentous fungi genomes which structure differs from most well annotated yeast genomes. Podospora anserina is a filamentous fungal model, which genome has been sequenced and annotated in 2008. Still, the current annotation lacks information about cis-regulatory elements, including promoters, transcription starting sites and terminators, which are instrumental to integrate epigenomic features into global gene regulation strategies.ResultsHere we took advantage of 37 RNA-seq experiments that were obtained in contrasted developmental and physiological conditions, to complete the functional annotation of P. anserina genome. Out of the 10,800 previously annotated genes, 5’UTR and 3’UTR were defined for 7554, among which, 3328 showed differential transcriptional signal starts and/or transcriptional end sites. In addition, alternative splicing events were detected for 2350 genes, mostly due alternative 3’splice sites and 1732 novel transcriptionally active regions (nTARs) in unannotated regions were identified.ConclusionsOur study provides a comprehensive genome-wide functional annotation of P. anserina genome, including chromatin features, cis-acting elements such as UTRs, alternative splicing events and transcription of non-coding regions. These new findings will likely improve our understanding of gene regulation strategies in compact genomes, such as those of filamentous fungi. Characterization of alternative transcripts and nTARs paves the way to the discovery of putative new genes, alternative peptides or regulatory non-coding RNAs.

BaRTv2: a highly resolved barley reference transcriptome for accurate transcript-specific RNA-seq quantification.

SUMMARYAccurate characterisation of splice junctions (SJs) as well as transcription start and end sites in reference transcriptomes allows precise quantification of transcripts from RNA‐seq data, and enables detailed investigations of transcriptional and post‐transcriptional regulation. Using novel computational methods and a combination of PacBio Iso‐seq and Illumina short‐read sequences from 20 diverse tissues and conditions, we generated a comprehensive and highly resolved barley reference transcript dataset from the European 2‐row spring barley cultivar Barke (BaRTv2.18). Stringent and thorough filtering was carried out to maintain the quality and accuracy of the SJs and transcript start and end sites. BaRTv2.18 shows increased transcript diversity and completeness compared with an earlier version, BaRTv1.0. The accuracy of transcript level quantification, SJs and transcript start and end sites have been validated extensively using parallel technologies and analysis, including high‐resolution reverse transcriptase‐polymerase chain reaction and 5'‐RACE. BaRTv2.18 contains 39 434 genes and 148 260 transcripts, representing the most comprehensive and resolved reference transcriptome in barley to date. It provides an important and high‐quality resource for advanced transcriptomic analyses, including both transcriptional and post‐transcriptional regulation, with exceptional resolution and precision.

Computational identification of signals predictive for nuclear RNA exosome degradation pathway targeting.

The RNA exosome degrades transcripts in the nucleoplasm of mammalian cells. Its substrate specificity is mediated by two adaptors: the ‘nuclear exosome targeting (NEXT)’ complex and the ‘poly(A) exosome targeting (PAXT)’ connection. Previous studies have revealed some DNA/RNA elements that differ between the two pathways, but how informative these features are for distinguishing pathway targeting, or whether additional genomic features that are informative for such classifications exist, is unknown. Here, we leverage the wealth of available genomic data and develop machine learning models that predict exosome targets and subsequently rank the features the models use by their predictive power. As expected, features around transcript end sites were most predictive; specifically, the lack of canonical 3′ end processing was highly predictive of NEXT targets. Other associated features, such as promoter-proximal G/C content and 5′ splice sites, were informative, but only for distinguishing NEXT and not PAXT targets. Finally, we discovered predictive features not previously associated with exosome targeting, in particular RNA helicase DDX3X binding sites. Overall, our results demonstrate that nucleoplasmic exosome targeting is to a large degree predictable, and our approach can assess the predictive power of previously known and new features in an unbiased way.

A high-resolution single-molecule sequencing-based Arabidopsis transcriptome using novel methods of Iso-seq analysis

BackgroundAccurate and comprehensive annotation of transcript sequences is essential for transcript quantification and differential gene and transcript expression analysis. Single-molecule long-read sequencing technologies provide improved integrity of transcript structures including alternative splicing, and transcription start and polyadenylation sites. However, accuracy is significantly affected by sequencing errors, mRNA degradation, or incomplete cDNA synthesis.ResultsWe present a new and comprehensive Arabidopsis thaliana Reference Transcript Dataset 3 (AtRTD3). AtRTD3 contains over 169,000 transcripts—twice that of the best current Arabidopsis transcriptome and including over 1500 novel genes. Seventy-eight percent of transcripts are from Iso-seq with accurately defined splice junctions and transcription start and end sites. We develop novel methods to determine splice junctions and transcription start and end sites accurately. Mismatch profiles around splice junctions provide a powerful feature to distinguish correct splice junctions and remove false splice junctions. Stratified approaches identify high-confidence transcription start and end sites and remove fragmentary transcripts due to degradation. AtRTD3 is a major improvement over existing transcriptomes as demonstrated by analysis of an Arabidopsis cold response RNA-seq time-series. AtRTD3 provides higher resolution of transcript expression profiling and identifies cold-induced differential transcription start and polyadenylation site usage.ConclusionsAtRTD3 is the most comprehensive Arabidopsis transcriptome currently. It improves the precision of differential gene and transcript expression, differential alternative splicing, and transcription start/end site usage analysis from RNA-seq data. The novel methods for identifying accurate splice junctions and transcription start/end sites are widely applicable and will improve single-molecule sequencing analysis from any species.

Bookend: precise transcript reconstruction with end-guided assembly

We developed Bookend, a package for transcript assembly that incorporates data from different RNA-seq techniques, with a focus on identifying and utilizing RNA 5′ and 3′ ends. We demonstrate that correct identification of transcript start and end sites is essential for precise full-length transcript assembly. Utilization of end-labeled reads present in full-length single-cell RNA-seq datasets dramatically improves the precision of transcript assembly in single cells. Finally, we show that hybrid assembly across short-read, long-read, and end-capture RNA-seq datasets from Arabidopsis thaliana, as well as meta-assembly of RNA-seq from single mouse embryonic stem cells, can produce reference-quality end-to-end transcript annotations.