Live-Cell Visualization of Pre-mRNA Splicing with Single-Molecule Sensitivity
Open accessAbstract
Removal of introns from pre-messenger RNAs (pre-mRNAs) via splicing provides a versatile means of genetic regulation that is often disrupted in human diseases. To decipher how splicing occurs in real time, we directly examined with single-molecule sensitivity the kinetics of intron excision from pre-mRNA in the nucleus of living human cells. By using two different RNA labeling methods, MS2 and λN, we show that β-globin introns are transcribed and excised in 20-30 s. Furthermore, we show that replacing the weak polypyrimidine (Py) tract in mouse immunoglobulin μ (IgM) pre-mRNA by a U-rich Py decreases the intron lifetime, thus providing direct evidence that splice-site strength influences splicing kinetics. We also found that RNA polymerase II transcribes at elongation rates ranging between 3 and 6 kb min(-1) and that transcription can be rate limiting for splicing. These results have important implications for a mechanistic understanding of cotranscriptional splicing regulation in the live-cell context.
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Other imaging techniques reported RNA Pol II speed between 0.6 and 12 kb/min.
“Other imaging techniques such as fluctuation analysis of fluorescently labeled nascent RNA or high‐speed 3D fluorescence nanoimaging techniques reported speed in the same range, albeit slightly higher between 0.6 and 12 kb/min (Larson et al, 2011; Martin et al, 2013; Annibale & Gratton, 2015).”
From RNA polymerase II speed: a key player in controlling and adapting transcriptome composition (2021) · cites this paper as Martin et al, 2013
The Results report an RNA polymerase II elongation rate of predominantly 3–6 kb/min, measured by live-cell fluorescence imaging. That falls within the citing sentence’s combined 0.6–12 kb/min range, but this paper does not report the range’s endpoints. (LLM, conf=0.86)
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References 59 · Cited by 173
- Authors
- Martin, R. M., Rino, J., Carvalho, C., Kirchhausen, T., & Carmo-Fonseca, M.
- Affiliations
- University of Lisbon (PT)
- Boston Children's Hospital (US)
- Corresponding author
- Kirchhausen, Tomas
- Venue
- Cell Reports
- Publisher
- Elsevier BV
- Published
- 2013-09
- Volume
- 4
- Issue
- 6
- Pages
- 1144-1155
- ISSN
- 2211-1247 (ISSN-L)
- PMC
- PMC3805459
- OpenAlex
- W2100794056
- Cited by
- 173
- References
- 59
- Citations by year
- 2026: 10 · 2025: 7 · 2024: 11 · 2023: 10 · 2022: 12 · 2021: 16 · 2020: 18 · 2019: 15 · 2018: 16 · 2017: 13 · 2016: 23 · 2015: 10 · 2014: 11 · 2013: 1
- FWCI
- 6.72
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- Cell Reports (publishedVersion, cc-by-nc-nd)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Portuguese National Funding Agency for Science, Research and Technology (RCAAP Project by FCT) (submittedVersion, other-oa)
- Funders
- National Institute of General Medical Sciences (GM075252, R01GM075252)
- National Institute of Allergy and Infectious Diseases (U54AI057159)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Research and Splicing
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA and protein synthesis mechanisms
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
- Keywords
- pre-mRNA splicing, intron excision, splicing kinetics, live-cell imaging, MS2, polypyrimidine tract, splice site strength, RNA polymerase II elongation, transcription rate, co-transcriptional splicing
- MeSH terms
- Animals, HeLa Cells, Humans, Introns, RNA Splicing, RNA, Messenger, RNA Splice Sites, Mice, beta-Globins
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