Nascent RNA analyses: tracking transcription and its regulation

Abstract

The programmes that direct an organism's development and maintenance are encoded in its genome. Decoding of this information begins with regulated transcription of genomic DNA into RNA. Although transcription and its control can be tracked indirectly by measuring stable RNAs, it is only by directly measuring nascent RNAs that the immediate regulatory changes in response to developmental, environmental, disease and metabolic signals are revealed. Multiple complementary methods have been developed to quantitatively track nascent transcription genome-wide at nucleotide resolution, all of which have contributed novel insights into the mechanisms of gene regulation and transcription-coupled RNA processing. Here we critically evaluate the array of strategies used for investigating nascent transcription and discuss the recent conceptual advances they have provided.

Figure 1 |. The transcription cycle.

a | Typical gene architecture depicting DNA elements that affect transcription and transcript stability. At promoters, binding sites for gene-specific transcription factors are found between two core promoters (i.e., core initiation regions) that drive divergent transcription. The coding strand (right) produces mRNA that is stabilized by the presence of splice sites, and the anti-sense strand (left) produces an unstable upstream antisense RNA. b | The transcription cycle consists of the following steps. 1) A pioneer transcription factor binds a specific sequence motif and increases chromatin accessibility. 2) Additional sequence-specific TFs bind near the pioneer factor. Core promoters recruit GTFs and Pol II to form the PIC. 3) The GTF TFIIH unwinds DNA, and Pol II initiates transcription. 4) Pol II undergoes promoter-proximal pausing after transcribing 20–60 nt, and the pause is stabilized by binding of DSIF and NELF. Before or during pausing, Pol II’s CTD is phosphorylated at Ser5 and Ser7, and the RNA undergoes 5’ capping. 5) Pol II escapes promoter-proximal pausing and enters productive elongation, largely due to CDK9 phosphorylating multiple targets: NELF (ending its interaction with Pol II), DSIF (converting it to an elongation factor), and Pol II Ser2 (which interacts with RNA processing factors). Additional elongation factors, such as PAF1, promote this escape. 6) During productive elongation, co-transcriptional processing, including splicing, RNA methylation, and RNA editing occur. Nucleosomes are removed in highly transcribed genes, while chromatin accessibility increases at moderately transcribed genes^,. 7) The RNA is cleaved and polyadenylated. After cleavage, Pol II continues elongating, but the nascent RNA lacks a cap and subject to XRN2-mediated degradation, which destabilizes Pol II and contributes to termination. After termination, Pol II can be recycled to new initiation, repeating the transcriptional cycle.

Figure 2 |. Comparison of nascent RNA enrichment and sequencing assays.

Transcription profiles of a highly expressed gene (β-actin), a highly paused gene (MED17), and a gene with a nearby eRNA (IPO7 for human cells, Klf4 for mouse cells), generated with distinct RNA-seq methodologies. a | In chromatin-associated RNA (caRNA) methods, high salt washes are used to isolate chromatin-bound RNAs. In traditional caRNA-seq (lower), that material is directly sequenced. Start-seq (upper) further enriches for capped caRNAs and uses size selection to capture initiation and pause sites of individual transcripts. Data for caRNA originates from Ref. 46 and Start-seq from Ref. 23. b | Immunoprecipitation of Pol II complexes enriches for RNAs that associate with Pol II in mNET-seq. Antibody-mediated isolation of Pol II removes most chromatin-bound RNAs. mNET-seq data that used an antibody targeting total Pol II was obtained from Ref. 61. . c | Run-on techniques mark nascent RNAs with labeled nucleotides. The use of the anionic detergent sarkosyl in the run-on reaction releases paused polymerases but not back-tracked or terminated Pol II. The original genome-wide nuclear run-on assay, GRO-seq, has been adapted to provide single-nucleotide resolution of the position of engaged Pol II on nascent RNA (PRO-seq). Using cap selection and sequencing from the 5’ end of the labeled RNA reports the initiating base (PRO-cap, upper). Data for PRO-cap from Ref. 209 (GSE110638), for PRO-seq from Ref. 4. d | Metabolic RNA labeling methods feed living cells modified ribonucleotides that will be incorporated into nascent RNAs. After labeling, nascent RNA can be enriched from the total RNA pool with immuno-purification (TT-seq, upper). Additionally, 4-thiouridine allows U to C base conversion for mutation-based identification of nascent transcripts after sequencing (TT-TimeLapse-seq, lower). Data for TT-seq from Ref. 210, for TimeLapse-seq from Ref. 76.

Figure 3 |. Imaging nascent RNA.

a | Nascent RNAs can be detected via fluorescence in situ hybridization. Dye-labeled probes that are complementary to RNA hybridize to intronic sequences, permitting detection of endogenous nascent transcripts. b | Hairpin-forming sequences that bind the GFP-tagged MS2 protein are engineered into an intron and can be imaged in vivo.

Figure 4 |. Promoter-proximal pausing interferes with transcription initiation.

a | Structural modeling shows that Pol II pausing within 50 nt of the initiation site precludes PIC binding due to steric hindrance. Reprinted from Ref. 70. b | Analysis of coPRO data demonstrates that promoter-proximal pausing occurs within 60 nt genome-wide. Pausing predominantly occurs one base upstream from a cytosine in a GC-rich region. Adapted from Ref. 52. c | Updated transcription cycle model demonstrating that paused Pol II blocks PIC formation.

Figure 5 |. Observing cleavage and polyadenylation in nascent RNA datasets.

The 3’ end of a gene is demarcated by a polyadenylation signal (PAS) that is slightly upstream of the cleavage and polyadenylation site (CPS). In typical cells, Pol II accumulates at the CPS, which is indicative of a reduced elongation rate. After cleavage, Pol II continues transcribing, but its levels decrease as termination occurs in a termination window. During cleavage factor knock down, Pol II accumulates far less at the PAS and terminates later. Adapted from Ref. 46. When the XRN2 protein is rapidly degraded, Pol II has slightly less accumulation at the CPS but very delayed termination. Adapted from Ref. 172. These results support the torpedo model, whereby XRN2 chases down unshielded nascent RNA after the cleavage, destabilizing Pol II.

Figure 6 |. Histone modifications correlate with transcription level.

Previous work suggests that H3K4me1 demarcates enhancers and H3K4me3 demarcates promoters. Nascent RNA experiments demonstrate that the degree of H3K4 methylation instead correlates with transcription level. Enhancers are commonly enriched for H3K4me1 because they have lower transcription than promoters.