Why Study Nascent RNA?

Nascent RNA refers to newly synthesized RNA molecules in cells, which are unstable and not yet fully processed. These transcripts provide a direct snapshot of a cell's transcriptional activity. Gene expression is a highly dynamic process, regulated by both intrinsic cellular programs and extrinsic environmental cues. Conventional RNA-seq captures only steady-state RNA levels and cannot discriminate newly synthesized nascent transcripts, limiting its ability to reveal the temporal dynamics of transcription. Nascent RNA sequencing addresses this limitation by directly labeling and enriching RNA that is undergoing synthesis, enabling kinetic analyses of transcriptional processes, such as initiation, pausing, elongation, and termination. This approach offers deeper mechanistic insights into gene regulation and provides novel perspectives for understanding disease processes and cellular response mechanisms.

Differences between PRO-seq, TT-seq, and conventional RNA-seq

Nascent RNA Enables Investigation of Transcriptional Kinetics
Conventional RNA-seq measures steady-state RNA levels, whereas nascent RNA sequencing provides a direct view of transcriptional kinetics. Among current methods for nascent RNA profiling, TT-seq (Transient Transcriptome Sequencing) and PRO-seq (Precision Run-On Sequencing) are widely used for their ability to capture immediate transcriptional dynamics.

1. TT-seq

TT-seq is a metabolic labeling-based nascent RNA sequencing technique. Cells are incubated with the nucleoside analog 4-thiouridine (4sU), which is incorporated into multiple uridine residues of nascent RNA within a defined labeling period. The labeled RNA fragments are subsequently enriched and sequenced. Unlike conventional RNA-seq, which reflects steady-state RNA levels, TT-seq provides high temporal resolution and quantitative sensitivity, enabling precise monitoring of transcriptional dynamics.

Applications

(1) Capturing Minute-Scale Transcriptional Dynamic Changes

TT-seq precisely captures dynamic transcriptional responses, making it suitable for studying rapid transcriptional events such as signaling pathway activation and drug stimulation.

TT-seq, combined with transcription inhibitors, enables resolution of transcription rates

(2) Detecting Expression Levels of Both Coding and Non-coding Nascent RNAs

TT-seq detects both coding and non-coding RNAs being synthesized, providing a comprehensive view of genome-wide transcriptional activity.

TT-seq enables analysis of both coding and non-coding nascent RNAs

(3) Analyzing Drug Action and Transcriptional Regulatory Mechanisms

TT-seq detects transient transcriptional changes induced by drug stimulation to analyze mechanisms of action.

Cluster analysis of differentially expressed genes in bone marrow macrophages following Mycobacterium tuberculosis infection shows that TT-seq reveals glyoxylate reprograms the host transcriptome to combat pathogen infection

(4) Studying Bidirectional Transcription Events

TT-seq directly detects transcriptional direction, enabling investigation of molecular mechanisms underlying bidirectional and divergent transcription at promoters and regulatory elements.

TT-seq enables investigation of transcription in both forward and reverse directions

2. PRO-seq

PRO-seq precisely captures ongoing transcription events in cells or frozen tissues with single-base resolution. It directly maps the immediate distribution of the transcriptional machinery on the genome through a one-step in vitro extension and enrichment. PRO-seq accurately records the earliest gene responses to environmental or drug stimuli on a minute scale, overcoming the limitations of conventional RNA-seq and ChIP-seq in detecting immediate transcriptional changes with insufficient resolution. The method features high resolution, high sensitivity, and high temporal resolution.

Applications

(1) Detecting Transcriptional Dynamics of Low-Abundance Transcripts

PRO-seq has high sensitivity, enabling detection of low-abundance transcripts such as eRNAs (enhancer RNAs) and transiently paused genes. It is suitable for identifying nascent transcripts in complex samples, including those from microorganisms.

PRO-seq detects transcription of E. coli small RNAs, including regions partially undetected by RNA-seq

(2) Detecting Transcription Pausing, Elongation, and Termination Events with High Resolution

During PRO-seq transcription elongation, only one biotin-labeled nucleotide is incorporated per transcript, allowing accurate mapping of transcription complexes and precise measurement of dynamics such as pausing and elongation.

PRO-seq heatmaps around pause sites for investigating mechanisms of premature transcription termination

(3) Analyzing Transcriptional Regulatory Mechanisms

PRO-seq is used to study dynamic transcriptional regulatory mechanisms and to identify key nascent transcripts within regulatory networks.

PRO-seq enables analysis of early termination and elongation defects of RNA polymerase II during INTS1 deletion

(4) Studying the Transcriptional Regulatory Role of Chromatin Modifications

PRO-seq can be integrated with chromatin modification profiles (e.g., H3K27me3) to analyze how chromatin states influence transcriptional regulation.

Prediction of histone modifications based on nascent transcriptome data

Technologically Advanced Methods for Nascent RNA Library Construction

BiOligo has launched TT-seq and PRO-seq nascent RNA library preparation kits, designed to address key technical challenges in studying gene expression dynamics. Based on an optimized capture and library construction system, these kits enable efficient, high-sensitivity detection of nascent RNA. They provide tools for real-time kinetic analysis and a one-stop solution for research in gene expression regulation, disease mechanisms, and drug target identification.

BiOligo TT-seq (left) and PRO-seq (right) Library Preparation Kits

Product Features

1. Integrated Capture and Library Construction Workflow

The kit integrates all reagents and steps for nascent RNA capture and library construction, eliminating the need for separate capture components and library adapters. It streamlines the library construction process, significantly reducing preparation time.

2. High Signal-to-Noise Ratio and Sensitivity

The kit comprehensively covers both high- and low-abundance transcripts, as well as long- and short-lived RNAs, with minimal background noise. It provides enhanced sensitivity for detecting low-abundance transcripts, such as eRNAs and transiently paused genes.

3. High-Quality Library Construction

An optimized enzymatic reaction system ensures high library yield and quality, with uniform sequencing coverage and high rates of data demultiplexing.

Quality control results for PRO-seq libraries

How to Choose Between TT-seq and PRO-seq?

TT-seq is suitable for studying transcription rates and overall nascent RNA levels, focusing on transcriptional output and dynamic changes. PRO-seq, in contrast, emphasizes the precise localization of RNA polymerase on the genome, reflecting transcriptional dynamics and immediate transcriptional activity, enabling detailed observation of transcription speed and different stages of the transcription process.

In practical research, TT-seq and PRO-seq can also be used in combination. TT-seq reveals global changes in nascent RNA expression, while PRO-seq provides detailed 

information on the transcription process and regulatory stages. This combined approach enables comprehensive analysis of nascent RNA expression characteristics, as well as the distribution and regulatory features of transcriptional changes across the genome.

Product Recommendations

Recommended Articles

Title: Rapid unleashing of macrophage efferocytic capacity via transcriptional pause release
Journal: Nature
Date: 2024/03/13

Abstract: During development, inflammation, or tissue injury, macrophages may successively engulf and process multiple apoptotic corpses via efferocytosis to achieve tissue homeostasis. How macrophages may rapidly adapt their transcription to achieve continuous corpse uptake is incompletely understood. Transcriptional pause/release is an evolutionarily conserved mechanism in which RNA polymerase (Pol) II initiates transcription for 20–60 nucleotides, is paused for minutes to hours, and is then released to make full-length mRNA. Here, we show that macrophages, within minutes of corpse encounter, use transcriptional pause/release to unleash a rapid transcriptional response. For human and mouse macrophages, the Pol II pause/release was required for continuous efferocytosis in vitro and in vivo. Interestingly, blocking Pol II pause/release did not impede Fc receptor-mediated phagocytosis, yeast uptake, or bacterial phagocytosis. Integration of data from three genomic approaches—precision nuclear run-on sequencing, RNA sequencing, and assay for transposase-accessible chromatin using sequencing (ATAC-seq)—on efferocytic macrophages at different time points revealed that Pol II pause/release controls expression of select transcription factors and downstream target genes. Mechanistic studies on transcription factor EGR3, prominently regulated by pause/release, uncovered EGR3-related reprogramming of other macrophage genes involved in cytoskeleton and corpse processing. Using lysosomal probes and a new genetic fluorescent reporter, we identify a role for pause/release in phagosome acidification during efferocytosis. Furthermore, microglia from egr3-deficient zebrafish embryos displayed reduced phagocytosis of apoptotic neurons and fewer maturing phagosomes, supporting defective corpse processing. Collectively, these data indicate that macrophages use Pol II pause/release as a mechanism to rapidly alter their transcriptional programs for efficient processing of the ingested apoptotic corpses and for successive efferocytosis.