Single-molecule nascent RNA sequencing identifies regulatory domain architecture at promoters and enhancers

Abstract Background The dynamic process of transcription termination produces transient RNA intermediates that are difficult to distinguish from each other via short-read sequencing methods. Results Here, 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. Conclusions Our method is applicable to establish a comprehensive termination landscape in a broad range of species.

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FLEP-seq: simultaneous detection of RNA polymerase II position, splicing status, polyadenylation site and poly(A) tail length at genome-wide scale by single-molecule nascent RNA sequencing

Nature Protocols ◽

10.1038/s41596-021-00581-7 ◽

2021 ◽

Author(s):

Yanping Long ◽

Jinbu Jia ◽

Weipeng Mo ◽

Xianhao Jin ◽

Jixian Zhai

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Rna Sequencing ◽

Single Molecule ◽

Simultaneous Detection ◽

Tail Length ◽

Polyadenylation Site ◽

Genome Wide ◽

Wide Scale ◽

Nascent Rna

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Faculty Opinions recommendation of Nascent RNA sequencing reveals distinct features in plant transcription.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.726838545.793526993 ◽

2017 ◽

Author(s):

Chentao Lin

Keyword(s):

Rna Sequencing ◽

Nascent Rna ◽

Distinct Features

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Single molecule real time (SMRT) full length RNA-sequencing reveals novel and distinct mRNA isoforms in human bone marrow cell subpopulations

10.1101/555326 ◽

2019 ◽

Cited By ~ 1

Author(s):

Anne Deslattes Mays ◽

Marcel O. Schmidt ◽

Garrett T. Graham ◽

Elizabeth Tseng ◽

Primo Baybayan ◽

...

Keyword(s):

Bone Marrow ◽

Real Time ◽

Rna Sequencing ◽

Single Molecule ◽

Marrow Cell ◽

Full Length ◽

Protein Isoforms ◽

Mrna Isoforms ◽

Transcript Isoforms ◽

Cell Subpopulations

AbstractHematopoietic cells are continuously replenished from progenitor cells that reside in the bone marrow. To evaluate molecular changes during this process, we analyzed the transcriptomes of freshly harvested human bone marrow progenitor (lineage-negative) and differentiated (lineage-positive) cells by single molecule, real time (SMRT) full length RNA sequencing. This analysis revealed a ∼5-fold higher number of transcript isoforms than previously detected and showed a distinct composition of individual transcript isoforms characteristic for bone marrow subpopulations. A detailed analysis of mRNA isoforms transcribed from the ANXA1 and EEF1A1 loci confirmed their distinct composition. The expression of proteins predicted from the transcriptome analysis was validated by mass spectrometry and validated previously unknown protein isoforms predicted e.g. for EEF1A1. These protein isoforms distinguished the lineage negative cell population from the lineage positive cell population. Finally, transcript isoforms expressed from paralogous gene loci (e.g. CFD, GATA2, HLA-A, B & C) also distinguished cell subpopulations but were only detectable by full length RNA sequencing. Thus, qualitatively distinct transcript isoforms from individual genomic loci separate bone marrow cell subpopulations indicating complex transcriptional regulation and protein isoform generation during hematopoiesis.

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Nanopore direct RNA sequencing maps the complexity of Arabidopsis mRNA processing and m6A modification

eLife ◽

10.7554/elife.49658 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 43

Author(s):

Matthew T Parker ◽

Katarzyna Knop ◽

Anna V Sherwood ◽

Nicholas J Schurch ◽

Katarzyna Mackinnon ◽

...

Keyword(s):

Rna Sequencing ◽

Single Molecule ◽

Tail Length ◽

Transcript Abundance ◽

Mrna Processing ◽

Full Length ◽

Transcription Start Sites ◽

Model Plant Arabidopsis Thaliana ◽

Defective Rna ◽

A Site

Understanding genome organization and gene regulation requires insight into RNA transcription, processing and modification. We adapted nanopore direct RNA sequencing to examine RNA from a wild-type accession of the model plant Arabidopsis thaliana and a mutant defective in mRNA methylation (m6A). Here we show that m6A can be mapped in full-length mRNAs transcriptome-wide and reveal the combinatorial diversity of cap-associated transcription start sites, splicing events, poly(A) site choice and poly(A) tail length. Loss of m6A from 3’ untranslated regions is associated with decreased relative transcript abundance and defective RNA 3′ end formation. A functional consequence of disrupted m6A is a lengthening of the circadian period. We conclude that nanopore direct RNA sequencing can reveal the complexity of mRNA processing and modification in full-length single molecule reads. These findings can refine Arabidopsis genome annotation. Further, applying this approach to less well-studied species could transform our understanding of what their genomes encode.

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A spatially restricted fibrotic niche in pulmonary fibrosis is sustained by M-CSF/M-CSFR signalling in monocyte-derived alveolar macrophages

European Respiratory Journal ◽

10.1183/13993003.00646-2019 ◽

2019 ◽

Vol 55 (1) ◽

pp. 1900646 ◽

Cited By ~ 30

Author(s):

Nikita Joshi ◽

Satoshi Watanabe ◽

Rohan Verma ◽

Renea P. Jablonski ◽

Ching-I Chen ◽

...

Keyword(s):

Pulmonary Fibrosis ◽

Single Cell ◽

Rna Sequencing ◽

Single Molecule ◽

Alveolar Macrophages ◽

Lineage Tracing ◽

Single Cell Rna Sequencing ◽

Pharmacological Blockade ◽

Macrophage Colony ◽

Druggable Targets

Ontologically distinct populations of macrophages differentially contribute to organ fibrosis through unknown mechanisms.We applied lineage tracing, single-cell RNA sequencing and single-molecule fluorescence in situ hybridisation to a spatially restricted model of asbestos-induced pulmonary fibrosis.We demonstrate that tissue-resident alveolar macrophages, tissue-resident peribronchial and perivascular interstitial macrophages, and monocyte-derived alveolar macrophages are present in the fibrotic niche. Deletion of monocyte-derived alveolar macrophages but not tissue-resident alveolar macrophages ameliorated asbestos-induced lung fibrosis. Monocyte-derived alveolar macrophages were specifically localised to fibrotic regions in the proximity of fibroblasts where they expressed molecules known to drive fibroblast proliferation, including platelet-derived growth factor subunit A. Using single-cell RNA sequencing and spatial transcriptomics in both humans and mice, we identified macrophage colony-stimulating factor receptor (M-CSFR) signalling as one of the novel druggable targets controlling self-maintenance and persistence of these pathogenic monocyte-derived alveolar macrophages. Pharmacological blockade of M-CSFR signalling led to the disappearance of monocyte-derived alveolar macrophages and ameliorated fibrosis.Our findings suggest that inhibition of M-CSFR signalling during fibrosis disrupts an essential fibrotic niche that includes monocyte-derived alveolar macrophages and fibroblasts during asbestos-induced fibrosis.

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Lung transplantation for patients with severe COVID-19

Science Translational Medicine ◽

10.1126/scitranslmed.abe4282 ◽

2020 ◽

Vol 12 (574) ◽

pp. eabe4282 ◽

Cited By ~ 1

Author(s):

Ankit Bharat ◽

Melissa Querrey ◽

Nikolay S. Markov ◽

Samuel Kim ◽

Chitaru Kurihara ◽

...

Keyword(s):

Respiratory Failure ◽

Pulmonary Fibrosis ◽

Lung Transplantation ◽

Single Cell ◽

Rna Sequencing ◽

Lung Tissue ◽

Single Molecule ◽

Sequencing Data ◽

Native Lung ◽

Single Cell Rna Sequencing

Lung transplantation can potentially be a life-saving treatment for patients with nonresolving COVID-19–associated respiratory failure. Concerns limiting lung transplantation include recurrence of SARS-CoV-2 infection in the allograft, technical challenges imposed by viral-mediated injury to the native lung, and the potential risk for allograft infection by pathogens causing ventilator-associated pneumonia in the native lung. Additionally, the native lung might recover, resulting in long-term outcomes preferable to those of transplant. Here, we report the results of lung transplantation in three patients with nonresolving COVID-19–associated respiratory failure. We performed single-molecule fluorescence in situ hybridization (smFISH) to detect both positive and negative strands of SARS-CoV-2 RNA in explanted lung tissue from the three patients and in additional control lung tissue samples. We conducted extracellular matrix imaging and single-cell RNA sequencing on explanted lung tissue from the three patients who underwent transplantation and on warm postmortem lung biopsies from two patients who had died from COVID-19–associated pneumonia. Lungs from these five patients with prolonged COVID-19 disease were free of SARS-CoV-2 as detected by smFISH, but pathology showed extensive evidence of injury and fibrosis that resembled end-stage pulmonary fibrosis. Using machine learning, we compared single-cell RNA sequencing data from the lungs of patients with late-stage COVID-19 to that from the lungs of patients with pulmonary fibrosis and identified similarities in gene expression across cell lineages. Our findings suggest that some patients with severe COVID-19 develop fibrotic lung disease for which lung transplantation is their only option for survival.

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Characterization of novel primary miRNA transcription units in human cells using Bru-seq nascent RNA sequencing

NAR Genomics and Bioinformatics ◽

10.1093/nargab/lqz014 ◽

2019 ◽

Vol 2 (1) ◽

Cited By ~ 1

Author(s):

Karan Bedi ◽

Michelle T Paulsen ◽

Thomas E Wilson ◽

Mats Ljungman

Keyword(s):

Rna Polymerase Ii ◽

Rna Sequencing ◽

Regulatory Networks ◽

Transcriptional Control ◽

Mature Mirnas ◽

Mirna Regulation ◽

Cell Type ◽

Data Resource ◽

Cell Type Specific ◽

Nascent Rna

Abstract MicroRNAs (miRNAs) are key contributors to gene regulatory networks. Because miRNAs are processed from RNA polymerase II transcripts, insight into miRNA regulation requires a comprehensive understanding of the regulation of primary miRNA transcripts. We used Bru-seq nascent RNA sequencing and hidden Markov model segmentation to map primary miRNA transcription units (TUs) across 32 human cell lines, allowing us to describe TUs encompassing 1443 miRNAs from miRBase and 438 from MirGeneDB. We identified TUs for 61 miRNAs with an unknown CAGE TSS signal for MirGeneDB miRNAs. Many primary transcripts containing miRNA sequences failed to generate mature miRNAs, suggesting that miRNA biosynthesis is under both transcriptional and post-transcriptional control. In addition to constitutive and cell-type specific TU expression regulated by differential promoter usage, miRNA synthesis can be regulated by transcription past polyadenylation sites (transcriptional read through) and promoter divergent transcription (PROMPTs). We identified 197 miRNA TUs with novel promoters, 97 with transcriptional read-throughs and 3 miRNA TUs that resemble PROMPTs in at least one cell line. The miRNA TU annotation data resource described here reveals a greater complexity in miRNA regulation than previously known and provides a framework for identifying cell-type specific differences in miRNA transcription in cancer and cell transition states.

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Nanopore device-based fingerprinting of RNA oligos and microRNAs enhanced with an Osmium tag

Scientific Reports ◽

10.1038/s41598-019-50459-8 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 3

Author(s):

Madiha Sultan ◽

Anastassia Kanavarioti

Keyword(s):

Solid State ◽

Nucleotide Sequence ◽

Nucleic Acids ◽

Rna Sequencing ◽

Single Molecule ◽

Selective Labeling ◽

Base Calling ◽

Alpha Hemolysin ◽

Oxford Nanopore ◽

Single Molecule Analysis

Abstract Protein and solid-state nanopores are used for DNA/RNA sequencing as well as for single molecule analysis. We proposed that selective labeling/tagging may improve base-to-base resolution of nucleic acids via nanopores. We have explored one specific tag, the Osmium tetroxide 2,2′-bipyridine (OsBp), which conjugates to pyrimidines and leaves purines intact. Earlier reports using OsBp-tagged oligodeoxyribonucleotides demonstrated proof-of-principle during unassisted voltage-driven translocation via either alpha-Hemolysin or a solid-state nanopore. Here we extend this work to RNA oligos and a third nanopore by employing the MinION, a commercially available device from Oxford Nanopore Technologies (ONT). Conductance measurements demonstrate that the MinION visibly discriminates oligoriboadenylates with sequence A15PyA15, where Py is an OsBp-tagged pyrimidine. Such resolution rivals traditional chromatography, suggesting that nanopore devices could be exploited for the characterization of RNA oligos and microRNAs enhanced by selective labeling. The data also reveal marked discrimination between a single pyrimidine and two consecutive pyrimidines in OsBp-tagged AnPyAn and AnPyPyAn. This observation leads to the conjecture that the MinION/OsBp platform senses a 2-nucleotide sequence, in contrast to the reported 5-nucleotide sequence with native nucleic acids. Such improvement in sensing, enabled by the presence of OsBp, may enhance base-calling accuracy in enzyme-assisted DNA/RNA sequencing.

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