Related Experiment Video
Updated: May 7, 2026
12:20
Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons
Published on: August 6, 2014
Live-cell visualization of pre-mRNA splicing with single-molecule sensitivity
Robert M Martin1, José Rino, Célia Carvalho
1Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, 1649-028 Lisboa, Portugal.
Cell Reports
|September 17, 2013
Summary
Researchers directly observed intron removal during pre-messenger RNA (pre-mRNA) splicing in living human cells. Splicing kinetics are influenced by splice-site strength and transcription rates, offering insights into gene regulation.
Area of Science:
- Molecular Biology
- Genetics
- Cell Biology
Background:
- Pre-messenger RNA (pre-mRNA) splicing is a critical genetic regulatory process often impaired in human diseases.
- Understanding the real-time kinetics of splicing is essential for deciphering its role in cellular function and disease.
- Previous studies lacked direct observation of splicing dynamics within living cells.
Purpose of the Study:
- To directly investigate the kinetics of intron excision from pre-mRNA in the nucleus of living human cells using single-molecule sensitivity.
- To determine the influence of splice-site strength on splicing kinetics.
- To assess the impact of transcription rates on the splicing process.
Main Methods:
- Employed two distinct RNA labeling techniques, MS2 and λN, for real-time observation.
- Utilized single-molecule sensitivity to monitor intron excision dynamics.
- Manipulated splice-site strength by altering the polypyrimidine tract in immunoglobulin μ (IgM) pre-mRNA.
Main Results:
- Demonstrated that β-globin introns are transcribed and excised within 20-30 seconds in living cells.
- Showed that a U-rich polypyrimidine tract significantly decreases intron lifetime, confirming splice-site strength's influence on splicing kinetics.
- Determined RNA polymerase II elongation rates between 3 and 6 kb/min and identified transcription as a potential rate-limiting step for splicing.
Conclusions:
- Provides direct, real-time evidence of intron excision kinetics in living human cells.
- Establishes a mechanistic link between splice-site strength, RNA polymerase II transcription rates, and cotranscriptional splicing regulation.
- Highlights the importance of these dynamics for understanding gene expression and human diseases.
Related Concept Videos
01:32RNA Splicing
Splicing is the process by which eukaryotic RNA is edited before its translation into protein. The RNA strand transcribed from eukaryotic DNA is called the primary transcript. The primary transcripts that become mRNAs are called precursor messenger RNAs (pre-mRNAs). Eukaryotic pre-mRNA contains alternating sequences of exons and introns. Exons are nucleotide sequences that code for proteins, whereas introns are the non-coding regions. In RNA splicing, introns are removed and exons are bonded...
01:32RNA Splicing
Splicing is the process by which eukaryotic RNA is edited before its translation into protein. The RNA strand transcribed from eukaryotic DNA is called the primary transcript. The primary transcripts that become mRNAs are called precursor messenger RNAs (pre-mRNAs). Eukaryotic pre-mRNA contains alternating sequences of exons and introns. Exons are nucleotide sequences that code for proteins, whereas introns are the non-coding regions. In RNA splicing, introns are removed and exons are bonded...
01:19Protein Dynamics in Living Cells
Different fluorescence-based techniques are used to study the protein dynamics in living cells. These techniques include FRAP, FRET, and PET.
Fluorescent recovery after photobleaching (FRAP) is a fluorescent-protein-based detection technique used to quantify protein movement rates within the cell. This method exposes a small portion of the cell to an intense laser beam. The laser beam causes permanent photobleaching of the fluorophore-tagged proteins in the exposed region. As the bleached...
Fluorescent recovery after photobleaching (FRAP) is a fluorescent-protein-based detection technique used to quantify protein movement rates within the cell. This method exposes a small portion of the cell to an intense laser beam. The laser beam causes permanent photobleaching of the fluorophore-tagged proteins in the exposed region. As the bleached...