Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2025:2863:207-222.
doi: 10.1007/978-1-0716-4176-7_13.

A Method for Rapid Inducible RNA Decay

Lauren A Blake  1   2 , Leslie Watkins  1   2 , Bin Wu  3   4   5
Affiliations

A Method for Rapid Inducible RNA Decay

Lauren A Blake et al. Methods Mol Biol. 2025.

Abstract

Modulating RNA decay is a powerful tool to investigate RNA degradation dynamics. Here, we describe a protocol to inducibly recruit protein factors to regulate target RNA metabolism, called Rapid Inducible Decay of RNA (RIDR). RIDR induces fast and synchronous decay of target mRNAs within minutes and enables direct visualization of mRNA decay dynamics and subcellular kinetics in living cells. Here, we provide detailed procedures to make stable cell lines, conduct fixed- and live-cell measurements, and perform data analysis. We discuss the potential pitfalls and make RIDR applicable to a general biology lab.

Keywords: Fluorescent in situ hybridization; Immunofluorescence; Kinetics; RNA decay; Single molecule imaging.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1
Rapid Inducible Decay of RNA (RIDR) system uses the FRB/FKBP/Rapamycin chemically inducible dimerization system to instigate RNA decay on demand for MS2-tagged transcripts
Fig. 2
Fig. 2
Timeline of procedures for fixed-cell and live-cell imaging to study RNA decay using RIDR
Fig. 3
Fig. 3
Hybridization chamber set-up
Fig. 4
Fig. 4
Knockdown of MBSv1 tagged ACTB mRNA. ACTB-MBS MEF + tet3G-RIDR cells were given no treatment (a) or treated with 100 nM rapamycin and 100 μM DRB for 2 h (b) and then smFISH-IF was performed as described in Subheading 3.5. Magenta = ACTB-MBS mRNA, Cyan = mGAPDH mRNA, Green = anti-DCP1a antibody for immunofluorescence. Scale bar is 5 μm for cell image and 2 μm for inset
See this image and copyright information in PMC

References

    1. Russo J, Heck AM, Wilusz J et al. (2017) Metabolic labeling and recovery of nascent RNA to accurately quantify mRNA stability. Methods 120:39–48. 10.1016/j.ymeth.2017.02.003 - DOI - PMC - PubMed
    1. Eulalio A, Behm-Ansmant I, Schweizer D et al. (2007) P-body formation is a consequence, not the cause, of RNA-mediated gene silencing. Mol Cell Biol 27:3970LP–3981LP. 10.1128/MCB.00128-07 - DOI - PMC - PubMed
    1. Wang C, Schmich F, Srivatsa S et al. (2018) Context-dependent deposition and regulation of mRNAs in P-bodies. elife 7:e29815. 10.7554/eLife.29815 - DOI - PMC - PubMed
    1. Courel M, Clément Y, Bossevain C et al. (2019) GC content shapes mRNA storage and decay in human cells. elife 8:e49708. 10.7554/eLife.49708 - DOI - PMC - PubMed
    1. Tutucci E, Livingston NM, Singer RH et al. (2018) Imaging mRNA in vivo, from birth to death. Annu Rev Biophys 47:85–106. 10.1146/annurev-biophys-070317-033037 - DOI - PubMed

LinkOut - more resources