Single-cell analysis of RNase L-mediated mRNA decay.
Methods Enzymol
; 692: 157-175, 2023.
Article
en En
| MEDLINE
| ID: mdl-37925178
ABSTRACT
Ribonuclease L (RNase L) is a mammalian endoribonuclease that initiates the mass degradation of cellular mRNAs in response to double-stranded RNA or viral infection. The kinetic rate of mRNA decay upon RNase L activation has been elusive because RNase L is heterogeneously activated with respect to time in individual cells. Herein, we describe a method using immunofluorescence combined with single-molecule fluorescence in situ hybridization (smFISH) to determine single-cell mRNA decay rates upon RNase L activation. Using these approaches, we deduce that the rate of mRNA decay upon RNase L activation is extremely rapid, whereby the half-life of stable mRNAs such as GAPDH mRNA is reduced to â¼15 minutes in individual cells. This allows for RNase L to degrade nearly every mRNA in a cell in less than 1 hour, which is much faster than the decay rate that would be derived using bulk measurement techniques for mRNA levels, such as qRT-PCR. These single-cell approaches can generally be employed to resolve mRNA decay kinetics in additional contexts.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Estabilidad del ARN
/
Endorribonucleasas
Límite:
Animals
Idioma:
En
Revista:
Methods Enzymol
Año:
2023
Tipo del documento:
Article
País de afiliación:
Estados Unidos