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Nascent RNA analyses: tracking transcription and its regulation

Nature Reviews Genetics volume 20, pages 705–723 (2019)Cite this article

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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.

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Fig. 1: The transcription cycle.
Fig. 2: Comparison of nascent RNA enrichment and sequencing assays.
Fig. 3: Imaging nascent RNA.
Fig. 4: Promoter-proximal pausing interferes with transcription initiation.
Fig. 5: Observing cleavage and polyadenylation in nascent RNA data sets.

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Acknowledgements

The authors thank members of the Lis laboratory for insightful discussions. They also thank the reviewers for their invaluable comments. This work was supported by the National Institute of General Medical Sciences grants R01-GM025232 (J.T.L.), F32-GM129904 (E.M.W.) and T32-HD057854 (N.D.T), and the Sigrid Jusélius Foundation (A.V.).

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  1. These authors contributed equally: Erin M. Wissink, Anniina Vihervaara.

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  1. Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, USA

    Erin M. Wissink, Anniina Vihervaara, Nathaniel D. Tippens & John T. Lis

  2. Tri-Institutional Training Program in Computational Biology and Medicine, New York, NY, USA

    Nathaniel D. Tippens

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Glossary

Enhancer RNAs

(eRNAs). Short (50–2000-nucleotide) non-coding RNAs that are produced by RNA polymerase II from enhancers. The production of enhancer RNAs moderately correlates with the functional activity of the enhancer.

Core initiation regions

Short (~60-nucleotide) regions at promoter and enhancer transcription start sites that provide a binding platform for general transcription factors. At promoters, ‘core promoter’ is used as a synonym for ‘core initiation region’.

C-terminal domain (CTD) of the RBP1 subunit of Pol II

This domain contains multiple repeats of seven amino acids and serves as a flexible binding scaffold for transcriptional regulators. The post-translational modifications of the heptad repeat greatly influence the regulatory interactions and, therefore, transcriptional processes throughout the cycle.

Nascent RNA

RNA that is in the process of being synthesized via transcription.

Divergent transcripts

In metazoans, genes and enhancers drive transcription from two core initiation regions from both strands in opposing directions. In genes, the mRNA-coding strand is termed the ‘sense strand’, and the antisense strand produces the divergent transcript.

Chromatin immunoprecipitation followed by sequencing (ChIP–seq)

A method in which protein–DNA interactions are stabilized, chromatin is sheared and fragments with a protein of interest are enriched using an antibody. Purified DNA from the enriched fragments is sequenced, providing genome-wide maps of protein localization.

RNA immunoprecipitation followed by sequencing

A technique that is used to identify which RNAs interact with a given protein. It uses antibody-mediated enrichment of a protein, after which its interacting RNAs can be isolated, reverse-transcribed and sequenced.

Paired-end sequencing

High-throughput sequencing of DNA molecules from both ends, which provides information from 3′ and 5′ ends of each DNA fragment, and allows more accurate mapping of the reads to the reference genome.

Permanganate treatment and chromatin immunoprecipitation followed by sequencing (PIP–seq)

A technique that uses permanganate, which oxidizes unpaired thymines in DNA, to detect the exact locations of open transcription complexes across the genome. RNA polymerase II is then immunoprecipitated, and DNA that has been oxidized by permanganate is cleaved using piperidine. These cleaved sites are identified by high-throughput sequencing.

Transcriptional bursting

Transcription occurs infrequently, and when a gene is turned on, many polymerases transcribe many copies of mRNA in a short time, which is known as transcriptional bursting. Bursting at a given gene is characterized by the duration, amplitude and frequency of transcription.

Cryogenic electron microscopy

An electron microscopy technique that visualizes molecules at cryogenic (−200 °C) temperatures. It allows near atomic resolution (less than 4 Å) imaging of complex molecules and molecule complexes in their native conformation without crystallization or embedding of the sample.

Intron definition

A splicing model in which specific sequences that demarcate introns are sufficient for spliceosomes to recognize intron boundaries.

Exon definition

A model by which proteins that bind to exons are required for the spliceosome to recognize sequences that demarcate introns.

R-loops

Three-stranded DNA–RNA hybrid structures formed, for example, by template DNA, the complement nascent RNA and the non-template single-stranded DNA.

Cap analysis of gene expression

A technique that measures RNA expression and maps transcription start sites of gene promoters. It provides precise maps of transcription start sites of genes that produce long-lived transcripts.

Self-transcribing active regulatory region sequencing (STARR-seq)

A method that assays enhancer activity for millions ofcandidate sequences by cloning them downstream of a reporter gene and upstream of a cleavage and polyadenylation site Functionally active enhancers drive expression of RNA molecules that contains the candidate sequence.

Degron system

A tool for rapidly degrading a specific protein in a cell. Genome editing is used to tag the protein of interest with a protein domain that is recognized by the E3 ubiquitin ligase complex. On addition of a small molecule, the tagged factor is inducibly degraded by the proteasome.

RNA aptamers

Structured RNA molecules selected for binding to a factor of interest to disrupt its functions or interactions.

Super-resolution microscopy

A collective term for light microscopy techniques that provide higher resolution (<200 nm) than imposed by the diffraction limit of visible light.

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Wissink, E.M., Vihervaara, A., Tippens, N.D. et al. Nascent RNA analyses: tracking transcription and its regulation. Nat Rev Genet 20, 705–723 (2019). https://doi.org/10.1038/s41576-019-0159-6

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