Gene expression is inherently dynamic. Conventional differential gene expression analysis (DRE) is difficult to capture transient changes and unstable non-coding RNAs (such as enhancer RNAs) in complex transcriptional regulation due to its technical limitations. In this context, RNA sequencing technology provides an important means for studying RNA dynamics by locating transcription start sites and quantifying nascent RNA.
However, compared with traditional DGE analysis, the study of nascent RNA faces more challenges, mainly due to its Short half-life and low abundance Characteristics. In recent years, a variety of analysis methods specifically for nascent RNA have emerged. These technologies reveal the degree of transcriptional activity in the promoter region, especially the residence time of PolⅡ in the proximal end of the promoter under the transcriptional activation state, which is a key link in gene expression regulation. In addition, nascent RNA can not only directly affect the transcription process, but its sequence and structural characteristics also regulate dynamic behaviors such as transcription elongation, pause and stagnation, and participate in the binding process of chromatin modifying enzymes and enhancer RNA. At present, nascent RNA-seq technologies aimed at distinguishing newly synthesized RNA from other RNAs are mainly divided into three categories: Transcription run-on based methods, PolⅡ immunoprecipitation (IP) based techniques, and metabolic labeling methods.
1. Run-on method
Enrichment of nascent RNA from total RNA by incorporation of nucleotide analogs during transcription and used to measure transient transcriptional activity (Figure a). Among them, Global run-on sequencing (GRO-seq) and Precision nuclear run-on sequencing (PRO-seq) use 5-bromouridine 5′-triphosphate (BrU) or biotin-labeled nucleotides to incorporate into nascent RNA, thereby locating the location and activity of active RNA polymerases within the transcriptome. In the experiment, after the cell nucleus is isolated, the endogenous nucleotides are washed away, and then exogenous labeled nucleotides are added to resume transcription. By enriching the nascent transcripts through immunoprecipitation or affinity chromatography, high-resolution detection of transcriptional activity can be achieved.
Due to the limitation of the amount of labeled nucleotide incorporation, GRO-seq has a resolution of only 10-50 bp, which affects the precise positioning of the transcription start site (TSS). In contrast, PRO-seq achieves single-base resolution positioning by terminating transcription after biotin nucleotide incorporation. Although the Run-on method is simple in concept (only RNA molecules incorporating modified nucleotides need to be enriched), the presence of background non-nascent RNA increases the read depth required for sequencing.
The application of these technologies has revealed the widespread existence of divergent or bidirectional transcription in promoter regions and clarified the role of enhancer RNA in gene expression regulation. In addition, by combining the specific enrichment of 5′-cap RNA (such as GRO-cap, PRO-cap or START-seq), the sensitivity and specificity of transcription initiation detection are further improved, while reducing the background signal interference caused by post-transcriptional capped RNA. These improved methods can also capture RNA that may be processed and removed during transcription, providing a more comprehensive perspective for studying transcription dynamics.
2. PolⅡ immunoprecipitation
Such as Native Elongating Transcription Sequencing (NET-seq) and its mammalian chromatin-modified version mNET-seq, Studying transcriptional dynamics by antibody-specific capture of Pol II-associated RNA These methods utilize anti-FLAG tag (against FLAG-tagged Pol II) or antibodies against the C-terminal domain of Pol II to enrich nascent RNA from chromatin complexes for mapping transcription start sites.
Although these techniques can directly correlate RNA polymerase activity with nascent RNA, they have the following limitations:
2.1 Background RNA interference:
Nonspecific binding of non-nascent RNA to Pol II and background mRNA will increase sequencing requirements and obscure analysis results.
2.2 Specificity issues:
NET-seq may enrich non-nascent RNAs (such as tRNA and small nucleolar RNAs) that strongly bind to PolⅡ, leading to data contamination.
2.3 Experimental complexity:
Although mNET-seq can reveal the regulatory mechanism of CTD modification and locate nascent RNA to TSS through a variety of CTD antibodies, it requires more cells, higher sequencing costs, and relies on complex experimental procedures.
3. Metabolic labeling
The nucleotide analog 4-thiouridine (4 sU) is used to label the nascent RNA, thereby enabling the detection of transcriptional dynamics (Figure c). However, methods with longer labeling times label the majority of transcripts, limiting sensitivity. Transient transcriptome sequencing (TT-seq) and thiol (SH)-linked alkylation RNA metabolic sequencing (SLAM-seq) address this problem by targeting the 3′ end of RNA (i.e., the newly transcribed region close to RNA polymerase), reducing background signals from 5′ RNA.
3.1 TT-seq:
Labeling time is limited to 5 minutes and only the 3′ end of new transcripts is labeled.
Add an RNA fragmentation step before biotin affinity purification to enrich the labeled RNA and improve detection sensitivity.
3.2 SLAM-seq:
Integrate 3' mRNA-seq library preparation (can also be used for miRNA libraries) to directly sequence tagged nascent RNAs rather than entire transcripts.
After RNA extraction, iodoacetamide is added to alkylate 4sU residues and induce reverse transcription-dependent T>C nucleotide conversion (manifested as a "mutation" in sequencing), thereby accurately locating the 4sU integration site.
However, the low integration rate results in only a few 4sU sites being converted to cytosine, limiting the detection sensitivity.
3.3 TUC-seq and TimeLapse-seq:
· The same T>C mutation analysis was used, but without enrichment of the 3' end.
Used to study transcriptional responses and RNA half-life measurements after cell perturbations.
To overcome the above difficulties, we developed a Click Chemistry Innovative nascent transcript capture and library construction kit for the reaction. The kit uses 5-Ethynyluracil (EU) The newly generated RNA in living cells is labeled, and then the newly generated RNA is labeled with biotin through a click chemistry reaction, and then the newly generated RNA is specifically captured by streptavidin magnetic beads, and finally the library is constructed for the newly generated RNA. Combined with the experimental time design, the newly generated RNA can be quantitatively analyzed, and the RNA transcription level and degradation issues can be studied at the same time.
For product details, please click: Nascent Transcript Kit
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