Population-scale study of eRNA transcription reveals bipartite functional enhancer architecture

AbstractEnhancer RNAs (eRNA) are unstable non-coding RNAs, transcribed bidirectionally from active regulatory sequences, whose expression levels correlate with enhancer activity. We use capped-nascent-RNA sequencing to efficiently capture bidirectional transcription initiation across several human lymphoblastoid cell lines (Yoruba population) and detect ~75,000 eRNA transcription sites with high sensitivity and specificity. The use of nascent-RNA sequencing sidesteps the confounding effect of eRNA instability. We identify quantitative trait loci (QTLs) associated with the level and directionality of eRNA expression. High-resolution analyses of these two types of QTLs reveal distinct positions of enrichment at the central transcription factor (TF) binding regions and at the flanking eRNA initiation regions, both of which are associated with mRNA expression QTLs. These two regions—the central TF-binding footprint and the eRNA initiation cores—define a bipartite architecture of enhancers, inform enhancer function, and can be used as an indicator of the significance of non-coding regulatory variants.

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Population-scale study of eRNA transcription reveals bipartite functional enhancer architecture

10.1101/426908 ◽

2018 ◽

Cited By ~ 5

Author(s):

Katla Kristjánsdóttir ◽

Yeonui Kwak ◽

Nathaniel D. Tippens ◽

John T. Lis ◽

Hyun Min Kang ◽

...

Keyword(s):

Transcription Initiation ◽

Large Scale ◽

High Sensitivity ◽

Regulatory Sequences ◽

Lymphoblastoid Cell Lines ◽

Human Lymphoblastoid Cell ◽

Regulatory Variants ◽

Transcription Sites ◽

Non Coding Rnas ◽

Human Lymphoblastoid Cell Lines

AbstractEnhancer RNAs (eRNA) are non-coding RNAs transcribed bidirectionally from active regulatory sequences. Their expression levels correlate with the activating potentials of the enhancers, but due to their instability, eRNAs have proven difficult to quantify in large scale. To overcome this, we use capped-nascent-RNA sequencing to efficiently capture the bidirectional initiation of eRNAs. We apply this in large scale to the human lymphoblastoid cell lines from the Yoruban population, and detected nearly 75,000 eRNA transcription sites with high sensitivity and specificity. We identify genetic variants significantly associated with overall eRNA initiation levels, as well as the transcription directionality between the two divergent eRNA pairs, namely the transcription initiation and directional initiation quantitative trait loci (tiQTLs and diQTLs) respectively. High-resolution analyses of these two types of eRNA QTLs reveal distinct positions of enrichment not only at the central transcription factor (TF) binding regions but also at the flanking eRNA initiation regions, both of which are equivalently associated with mRNA expression QTLs. These two regions - the central TF binding footprint and the eRNA initiation cores - define the bipartite architecture and the function of enhancers, and may provide further insights into interpreting the significance of non-coding regulatory variants.

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Landscape of transcription termination in Arabidopsis revealed by single-molecule nascent RNA sequencing

Genome Biology ◽

10.1186/s13059-021-02543-4 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Weipeng Mo ◽

Bo Liu ◽

Hong Zhang ◽

Xianhao Jin ◽

Dongdong Lu ◽

...

Keyword(s):

Rna Sequencing ◽

Single Molecule ◽

Transcription Initiation ◽

Transcription Termination ◽

Trna Genes ◽

Genome Wide ◽

Chimeric Transcripts ◽

Wide Range ◽

Wide Scale ◽

Nascent Rna

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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A Unified Probabilistic Modeling Framework for Eukaryotic Transcription Based on Nascent RNA Sequencing Data

10.1101/2021.01.12.426408 ◽

2021 ◽

Author(s):

Adam Siepel

Keyword(s):

Rna Sequencing ◽

Likelihood Ratio ◽

Transcription Initiation ◽

Likelihood Ratio Tests ◽

Parameter Estimates ◽

Sequencing Data ◽

Modeling Framework ◽

Library Size ◽

Eukaryotic Transcription ◽

Nascent Rna

AbstractNascent RNA sequencing protocols, such as GRO-seq and PRO-seq, are now widely used in the study of eukaryotic transcription, and these experimental techniques have given rise to a variety of statistical and machine-learning methods for data analysis. These computational methods, however, are generally designed to address specialized signal-processing or prediction tasks, rather than directly describing the dynamics of RNA polymerases as they move along the DNA template. Here, I introduce a general probabilistic model that describes the kinetics of transcription initiation, elongation, pause release, and termination, as well as the generation of sequencing read counts. I show that this generative model enables estimation of separate rates of initiation, pause-release, and termination, up to a proportionality constant. Furthermore, if applied to time-course data in a nonequilibrium setting, the model can be used to estimate elongation rates. This model additionally leads naturally to likelihood ratio tests for differences between genes, conditions, or species in various rates of interest. A version of the model in which read counts are assumed to be Poisson-distributed leads to convenient, closed-form solutions for parameter estimates and likelihood ratio tests. I present extensions to Bayesian inference and to a generalized linear model that can be used to discover genomic features associated with rates of elongation. Finally, I address technicalities concerning estimation of library size, normalization and sequencing replicates. Altogether, this modeling framework enables a unified treatment of many common tasks in the analysis of nascent RNA sequencing data.

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MicroRNAs in Human Lymphoblastoid Cell Lines

Critical Reviews in Eukaryotic Gene Expression ◽

10.1615/critreveukargeneexpr.v22.i3.20 ◽

2012 ◽

Vol 22 (3) ◽

pp. 189-196 ◽

Cited By ~ 8

Author(s):

Sung-Mi Shim ◽

Hye-Young Nam ◽

Jae-Eun Lee ◽

Jun-Woo Kim ◽

Bok-Ghee Han ◽

...

Keyword(s):

Cell Lines ◽

Lymphoblastoid Cell Lines ◽

Human Lymphoblastoid Cell ◽

Human Lymphoblastoid Cell Lines

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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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Spatial expression pattern of serine proteases in the blood fluke Schistosoma mansoni determined by fluorescence RNA in situ hybridization

Parasites & Vectors ◽

10.1186/s13071-021-04773-8 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Lenka Ulrychová ◽

Pavel Ostašov ◽

Marta Chanová ◽

Michael Mareš ◽

Martin Horn ◽

...

Keyword(s):

In Situ Hybridization ◽

Schistosoma Mansoni ◽

Rna Sequencing ◽

Serine Proteases ◽

Expression Patterns ◽

High Sensitivity ◽

Host Parasite Interactions ◽

Highly Sensitive ◽

Host Parasite

Abstract Background The blood flukes of genus Schistosoma are the causative agent of schistosomiasis, a parasitic disease that infects more than 200 million people worldwide. Proteases of schistosomes are involved in critical steps of host–parasite interactions and are promising therapeutic targets. We recently identified and characterized a group of S1 family Schistosoma mansoni serine proteases, including SmSP1 to SmSP5. Expression levels of some SmSPs in S. mansoni are low, and by standard genome sequencing technologies they are marginally detectable at the method threshold levels. Here, we report their spatial gene expression patterns in adult S. mansoni by the high-sensitivity localization assay. Methodology Highly sensitive fluorescence in situ RNA hybridization (FISH) was modified and used for the localization of mRNAs encoding individual SmSP proteases (including low-expressed SmSPs) in tissues of adult worms. High sensitivity was obtained due to specifically prepared tissue and probes in combination with the employment of a signal amplification approach. The assay method was validated by detecting the expression patterns of a set of relevant reference genes including SmCB1, SmPOP, SmTSP-2, and Sm29 with localization formerly determined by other techniques. Results FISH analysis revealed interesting expression patterns of SmSPs distributed in multiple tissues of S. mansoni adults. The expression patterns of individual SmSPs were distinct but in part overlapping and were consistent with existing transcriptome sequencing data. The exception were genes with significantly low expression, which were also localized in tissues where they had not previously been detected by RNA sequencing methods. In general, SmSPs were found in various tissues including reproductive organs, parenchymal cells, esophagus, and the tegumental surface. Conclusions The FISH-based assay provided spatial information about the expression of five SmSPs in adult S. mansoni females and males. This highly sensitive method allowed visualization of low-abundantly expressed genes that are below the detection limits of standard in situ hybridization or by RNA sequencing. Thus, this technical approach turned out to be suitable for sensitive localization studies and may also be applicable for other trematodes. The results suggest that SmSPs may play roles in diverse processes of the parasite. Certain SmSPs expressed at the surface may be involved in host–parasite interactions. Graphic abstract

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Small RNA-Sequencing: Approaches and Considerations for miRNA Analysis

Diagnostics ◽

10.3390/diagnostics11060964 ◽

2021 ◽

Vol 11 (6) ◽

pp. 964

Author(s):

Sarka Benesova ◽

Mikael Kubista ◽

Lukas Valihrach

Keyword(s):

Rna Sequencing ◽

Small Rna ◽

High Sensitivity ◽

Small Rna Sequencing ◽

Rna Seq ◽

Liquid Biopsies ◽

Comprehensive Overview ◽

Rna Molecules ◽

Novel Mirna ◽

The Many

MicroRNAs (miRNAs) are a class of small RNA molecules that have an important regulatory role in multiple physiological and pathological processes. Their disease-specific profiles and presence in biofluids are properties that enable miRNAs to be employed as non-invasive biomarkers. In the past decades, several methods have been developed for miRNA analysis, including small RNA sequencing (RNA-seq). Small RNA-seq enables genome-wide profiling and analysis of known, as well as novel, miRNA variants. Moreover, its high sensitivity allows for profiling of low input samples such as liquid biopsies, which have now found applications in diagnostics and prognostics. Still, due to technical bias and the limited ability to capture the true miRNA representation, its potential remains unfulfilled. The introduction of many new small RNA-seq approaches that tried to minimize this bias, has led to the existence of the many small RNA-seq protocols seen today. Here, we review all current approaches to cDNA library construction used during the small RNA-seq workflow, with particular focus on their implementation in commercially available protocols. We provide an overview of each protocol and discuss their applicability. We also review recent benchmarking studies comparing each protocol’s performance and summarize the major conclusions that can be gathered from their usage. The result documents variable performance of the protocols and highlights their different applications in miRNA research. Taken together, our review provides a comprehensive overview of all the current small RNA-seq approaches, summarizes their strengths and weaknesses, and provides guidelines for their applications in miRNA research.

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