Live-Cell Visualization of Pre-mRNA Splicing with Single-Molecule Sensitivity

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Abstract

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 canbe 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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APA

Martin, R. M., Rino, J., Carvalho, C., Kirchhausen, T., & Carmo-Fonseca, M. (2013). Live-Cell Visualization of Pre-mRNA Splicing with Single-Molecule Sensitivity. Cell Reports, 4(6), 1144–1155. https://doi.org/10.1016/j.celrep.2013.08.013

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