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. 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.

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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
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