In eukaryotic cells, RNA polymerase II synthesizes mRNA in three stages, initiation, elongation, and termination, and numerous factors determine how quickly a gene is transcribed to produce mRNA molecules through these steps. However, there are few techniques available to measure the rate of each step in living cells, which prevents a better understanding of transcriptional regulation. Here, we present a quantitative analysis method to extract kinetic rates of transcription from time-lapse imaging data of fluorescently labeled mRNA in live cells. Using embryonic fibroblasts cultured from two knock-in mouse models, we monitored transcription of β-actin and Arc mRNA labeled with MS2 and PP7 stem–loop systems, respectively. After inhibiting transcription initiation, we measured the elongation rate and the termination time by fitting the time trace of transcription intensity with a mathematical model function. We validated our results by comparing them with those from an autocorrelation analysis and stochastic simulations. This live-cell transcription analysis method will be useful for studying the regulation of elongation and termination steps, providing insight into the diverse mechanisms of transcriptional processes.
Bibliographical noteFunding Information:
This work was supported by the Creative-Pioneering Researchers Program through Seoul National University, Howard Hughes Medical Institute (HHMI)-Wellcome International Research Scholar Award from the Wellcome Trust (208468/Z/17/Z), and the Basic Science Research Program through the National Research Foundation of Korea (NRF) grant funded by the Korean government (2020R1A2C2007285). For the purpose of open access, the author has applied a CC BY public copyright license to any Author Accepted Manuscript version arising from this submission.
Copyright © 2022 Choi, Lee and Park.
- live-cell imaging
- single-cell analysis
- transcription elongation
- transcription termination