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Project description:BackgroundThe dynamic process of transcription termination produces transient RNA intermediates that are difficult to distinguish from each other via short-read sequencing methods.ResultsHere, we use single-molecule nascent RNA sequencing to characterize the various forms of transient RNAs during termination at genome-wide scale in wildtype Arabidopsis and in atxrn3, fpa, and met1 mutants. Our data reveal a wide range of termination windows among genes, ranging from ~ 50 nt to over 1000 nt. We also observe efficient termination before downstream tRNA genes, suggesting that chromatin structure around the promoter region of tRNA genes may block pol II elongation. 5' Cleaved readthrough transcription in atxrn3 with delayed termination can run into downstream genes to produce normally spliced and polyadenylated mRNAs in the absence of their own transcription initiation. Consistent with previous reports, we also observe long chimeric transcripts with cryptic splicing in fpa mutant; but loss of CG DNA methylation has no obvious impact on termination in the met1 mutant.ConclusionsOur method is applicable to establish a comprehensive termination landscape in a broad range of species.
| S-EPMC8613925 | biostudies-literature
Project description:The dynamic process of transcription termination produces transient RNA intermediates, such as the full-length readthrough, 5’ and 3’ cleavage products, and polyadenylated nascent RNAs, which are difficult to distinguish from each other via short-read sequencing methods. Here, we used single-molecule nascent RNA sequencing to characterize these various forms of transient RNAs during termination at genome-wide scale in Arabidopsis. Our data revealed a wide range of termination windows (defined as the median readthrough distance) among genes, ranging from ~50 nt to over 1000 nt. The ends of termination windows precisely match the peak of Pol II occupancy and co-localize with termination factors. We also discovered a termination mechanism shaped by the downstream tRNA genes, suggesting that elongating Pol II can be expelled by Pol III activity. Mutation of the exonuclease AtXRN3 leads to a drastic accumulation of 3’ cleaved readthrough RNAs. Moreover, readthrough transcription in atxrn3 with delayed termination can run into downstream genes to produce normally spliced and polyadenylated mRNAs in the absence of their own transcription initiation. Our results revealed the diverse termination patterns in Arabidopsis, and our method is applicable to study the global termination landscape in a broad range of species.
2024-09-22 | GSE184099 | GEO
Project description:The dynamic process of transcription termination produces transient RNA intermediates, such as the full-length readthrough, 5’ and 3’ cleavage products, and polyadenylated nascent RNAs, which are difficult to distinguish from each other via short-read sequencing methods. Here, we used single-molecule nascent RNA sequencing to characterize these various forms of transient RNAs during termination at genome-wide scale in Arabidopsis. Our data revealed a wide range of termination windows (defined as the median readthrough distance) among genes, ranging from ~50 nt to over 1000 nt. The ends of termination windows precisely match the peak of Pol II occupancy and co-localize with termination factors. We also discovered a termination mechanism shaped by the downstream tRNA genes, suggesting that elongating Pol II can be expelled by Pol III activity. Mutation of the exonuclease AtXRN3 leads to a drastic accumulation of 3’ cleaved readthrough RNAs. Moreover, readthrough transcription in atxrn3 with delayed termination can run into downstream genes to produce normally spliced and polyadenylated mRNAs in the absence of their own transcription initiation. Our results revealed the diverse termination patterns in Arabidopsis, and our method is applicable to study the global termination landscape in a broad range of species.
2024-09-22 | GSE178980 | GEO
Project description:Landscape of Transcription Termination in Arabidopsis Revealed by Single-molecule Nascent RNA Sequencing
| PRJNA763181 | ENA
Project description:Landscape of Transcription Termination in Arabidopsis Revealed by Single-molecule Nascent RNA Sequencing [FLEP-seq]
| PRJNA741784 | ENA
Project description:Landscape of Transcription Termination in Arabidopsis Revealed by Single-molecule Nascent RNA Sequencing [BS-seq]
| PRJNA763183 | ENA
Project description:Transcription is the primary regulatory step in gene expression. Divergent transcription initiation from promoters and enhancers produces stable RNAs from genes and unstable RNAs from enhancers1,2. Nascent RNA capture and sequencing assays simultaneously measure gene and enhancer activity in cell populations3. However, fundamental questions about the temporal regulation of transcription and enhancer-gene coordination remain unanswered, primarily because of the absence of a single-cell perspective on active transcription. In this study, we present scGRO-seq-a new single-cell nascent RNA sequencing assay that uses click chemistry-and unveil coordinated transcription throughout the genome. We demonstrate the episodic nature of transcription and the co-transcription of functionally related genes. scGRO-seq can estimate burst size and frequency by directly quantifying transcribing RNA polymerases in individual cells and can leverage replication-dependent non-polyadenylated histone gene transcription to elucidate cell cycle dynamics. The single-nucleotide spatial and temporal resolution of scGRO-seq enables the identification of networks of enhancers and genes. Our results suggest that the bursting of transcription at super-enhancers precedes bursting from associated genes. By imparting insights into the dynamic nature of global transcription and the origin and propagation of transcription signals, we demonstrate the ability of scGRO-seq to investigate the mechanisms of transcription regulation and the role of enhancers in gene expression.
| S-EPMC11222150 | biostudies-literature
Project description:Extrinsic transcription termination typically involves remodeling of RNA polymerase by an accessory helicase. In yeast this is accomplished by the Sen1 helicase homologous to human senataxin (SETX). To gain insight into these processes we develop a DNA scaffold construct compatible with magnetic-trapping assays and from which S. cerevisiae RNA polymerase II (Pol II), as well as E. coli RNA polymerase (ecRNAP), can efficiently initiate transcription without transcription factors, elongate, and undergo extrinsic termination. By stalling Pol II TECs on the construct we can monitor Sen1-induced termination in real-time, revealing the formation of an intermediate in which the Pol II transcription bubble appears half-rewound. This intermediate requires ~40 sec to form and lasts ~20 sec prior to final dissociation of the stalled Pol II. The experiments enabled by the scaffold construct permit detailed statistical and kinetic analysis of Pol II interactions with a range of cofactors in a multi-round, high-throughput fashion.
| S-EPMC6449345 | biostudies-literature
Project description:Eukaryotic RNA polymerase II (Pol II) has been found at both promoters and distal enhancers, suggesting additional functions beyond mRNA production. To understand this role, we sequenced nascent RNAs at single-molecule resolution to unravel the interplay between Pol II initiation, capping and pausing genome-wide. Our analyses identify two pause classes that are associated with different RNA capping profiles. More proximal pausing is associated with less complete capping, less elongation and a more enhancer-like complement of transcription factors than later pausing. Unexpectedly, transcription start sites (TSSs) are predominantly found in constellations composed of multiple divergent pairs. TSS clusters are intimately associated with precise arrays of nucleosomes and correspond with boundaries of transcription factor binding and chromatin modification at promoters and enhancers. TSS architecture is largely unchanged during the dramatic transcriptional changes induced by heat shock. Together, our results suggest that promoter- and enhancer-associated Pol II is a regulatory nexus for integrating information across TSS ensembles.
| S-EPMC6422046 | biostudies-literature
Project description:Temperature profoundly affects the kinetics of biochemical reactions, yet how large molecular complexes such as the transcription machinery accommodate changing temperatures to maintain cellular function is poorly understood. Here, we developed plant native elongating transcripts sequencing (plaNET-seq) to profile genome-wide nascent RNA polymerase II (RNAPII) transcription during the cold-response of Arabidopsis thaliana with single-nucleotide resolution. Combined with temporal resolution, these data revealed transient genome-wide reprogramming of nascent RNAPII transcription during cold, including characteristics of RNAPII elongation and thousands of non-coding transcripts connected to gene expression. Our results suggest a role for promoter-proximal RNAPII stalling in predisposing genes for transcriptional activation during plant-environment interactions. At gene 3'-ends, cold initially facilitated transcriptional termination by limiting the distance of read-through transcription. Within gene bodies, cold reduced the kinetics of co-transcriptional splicing leading to increased intragenic stalling. Our data resolved multiple distinct mechanisms by which temperature transiently altered the dynamics of nascent RNAPII transcription and associated RNA processing, illustrating potential biotechnological solutions and future focus areas to promote food security in the context of a changing climate.
| S-EPMC7049701 | biostudies-literature