Quantitative Imaging of MS2-Tagged hTR in Cajal Bodies: Photobleaching and Photoactivation

• Published in: STAR protocols Vol. 1; no. 3; p. 100112
• Main Authors: Smith, Michael, Querido, Emmanuelle, Chartrand, Pascal, Sfeir, Agnel
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 18.12.2020; Elsevier
• Subjects: ADAM Proteins - genetics; ADAM Proteins - metabolism; Coiled Bodies - metabolism; HeLa Cells; Humans; In Situ Hybridization, Fluorescence - methods; Inverted Repeat Sequences - genetics; Membrane Proteins - genetics; Membrane Proteins - metabolism; Photobleaching; Protocol; RNA - analysis; RNA - genetics; Single Molecule Imaging - methods; Telomerase - analysis; Telomerase - genetics; Telomere - metabolism
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2020.100112

Advances in imaging technologies, gene editing, and fluorescent molecule development have made real-time imaging of nucleic acids practical. Here, we detail methods for imaging the human telomerase RNA template, hTR via the use of three inserted MS2 stem loops and cognate MS2 coat protein (MCP) tagged with superfolder GFP or photoactivatable GFP. These technologies enable tracking of the dynamics of RNA species through Cajal bodies and offer insight into their residence time in Cajal bodies through photobleaching and photoactivation experiments. For complete details on the use and execution of this protocol, please refer to Laprade et al. (2020). [Display omitted] •Live-cell imaging allows visualization of telomerase RNA at the Cajal body in real time•FRAP analysis is useful for measuring entry of telomerase RNA into the Cajal body•Photoactivation analysis permits measurement of exit dynamics from the same structures•Culture conditions, analysis, and live-cell imaging considerations are detailed Advances in imaging technologies, gene editing, and fluorescent molecule development have made real-time imaging of nucleic acids practical. Here, we detail methods for imaging the human telomerase RNA template, hTR via the use of three inserted MS2 stem loops and cognate MS2 coat protein (MCP) tagged with superfolder GFP or photoactivatable GFP. These technologies enable tracking of the dynamics of RNA species through Cajal bodies and offer insight into their residence time in Cajal bodies through photobleaching and photoactivation experiments.