Transcriptional Pulsing Approaches for Analysis of mRNA Turnover in Mammalian Cells

doi:10.1006/meth.1998.0702

Methods

Volume 17, Issue 1, January 1999, Pages 11-20

https://doi.org/10.1006/meth.1998.0702 Get rights and content

Abstract

Modulation of mRNA stability provides a powerful means for controlling gene expression during the cell cycle, cell differentiation, the immune response, as well as many other physiological transitions. Through the years, many different methods have been developed for measuring mRNA stability. Frequently mRNA stability is studied indirectly by analyzing the steady-state level of mRNA. Therefore by inference, changes in mRNA abundance are thought to affect only the stability of the mRNA, an assumption that is not always correct. Alternatively, direct measurements of mRNA decay are performed in a number of ways, including kinetic labeling techniques and administration of transcriptional inhibitors. Due to the nature of these techniques, they either are technically demanding or introduce a significant change in cell physiology. In addition, many critical mechanistic issues as to deadenylation kinetics, decay intermediates, and precursor–product relationships cannot be readily addressed by these methods. Here, we describe and discuss in detail two different transcriptional pulsing methods based on the c-fosserum-inducible promoter and the tetracycline-regulated promoter systems as an effort to better elucidate the mechanistic steps and regulation underlying differential and selective mRNA turnover in mammalian cells. Both systems allow unequivocal monitoring of deadenylation and decay kinetics as well as determination of precursor–product relationship. In addition, decay rate constants and half-lives are determined and used in both methods to quantitatively denote the mRNA stability. Thus, they provide a reliable way to determine subtle yet physiologically meaningful changes in mRNA stability. Application of one method or the other covers the study of mRNA turnover in most mammalian cell types under a wide range of physiological conditions.

References (38)

C.A. Beelman et al.
Cell
(1995)
S. Harrold et al.
Anal. Biochem.
(1991)
C.-Y.A. Chen et al.
Cell
(1988)
C. Speth et al.
Exp. Cell Res.
(1993)
D.X. Yin et al.
Anal. Biochem.
(1996)
R. Treisman
Cell
(1985)
F.C. Nielsen et al.
J. Biol. Chem.
(1992)
G. Shaw et al.
Cell
(1986)
T.J. Ernst et al.
J. Biol. Chem.
(1989)
A. Jacobson et al.
Annu. Rev. Biochem.
(1996)

J. Ross

Microbiol. Rev.

(1995)

A.C.-Y. Chen et al.

Trends Biochem. Sci.

(1995)

J. Belasco et al.

Control of Messenger RNA Stability

(1993)

A.-B. Shyu et al.

Genes Dev.

(1989)

C.Y. Chen et al.

Mol. Cell. Biol.

(1995)

D. Muhlrad et al.

Genes Dev.

(1992)

A.-B. Shyu et al.

The Immunology Methods Manual

(1996)

C.Y. Chen et al.

Mol. Cell. Biol.

(1994)

Cited by (67)

Trick or TREAT: A Scary-Good New Approach for Single-Molecule mRNA Decay Analysis
2017, Molecular Cell
Renal Ammonium Ion Production and Excretion
2013, Seldin and Geibisch's The Kidney
Renal Ammonium Ion Production and Excretion
2012, Seldin and Giebisch's The Kidney: Physiology and Pathophysiology
Chapter 24 Assays for Determining Poly(A) Tail Length and the Polarity of mRNA Decay in Mammalian Cells
2008, Methods in Enzymology
This chapter describes several methods for measuring the length of the mRNA poly(A) tail and a novel method for measuring mRNA decay. Three methods for measuring the length of a poly(A) tail are presented: the poly(A) length assay, the ligation‐mediated poly(A) test (LM‐PAT), and the RNase H assay. The first two methods are PCR‐based assays involving cDNA synthesis from an oligo(dT) primer. The third method involves removing the poly(A) tail from the mRNA of interest. A major obstacle to studying the enzymatic step of mammalian mRNA decay has been the inability to capture mRNA decay intermediates with structural impediments such as the poly(G) tract used in yeast. To overcome this, we combined a standard kinetic analysis of mRNA decay with a tetracycline repressor–controlled reporter with an Invader^® RNA assay. The Invader RNA assay is a simple, elegant assay for the quantification of mRNA. It is based on signal amplification, not target amplification, so it is less prone to artifacts than other methods for nucleic acid quantification. It is also very sensitive, able to detect attomolar levels of target mRNA. Finally, it requires only a short sequence for target recognition and quantitation. Therefore, it can be applied to determining the decay polarity of a mRNA by measuring the decay rates of different portions of that mRNA.
Chapter 17 Messenger RNA Half-Life Measurements in Mammalian Cells
2008, Methods in Enzymology
Citation Excerpt :
After transient transfection of the Tet‐off promoter–driven reporter plasmid, the transfected cells should be kept in culture medium containing the identified concentration of tetracycline until transcription induction (transcriptional pulsing). It is striking that all three different mammalian stable lines we established, including mouse NIH3T3 B2A2, human K562 III‐2, and human BEAS‐2B‐19, show the same transcriptional pulsing kinetics in Northern blot analysis (Chen et al., 2007; Loflin et al., 1999b; Xu et al., 1998). A population of mRNA homogenous in size appears in the cytoplasm after removal of tetracycline for 100 to 110 min, making it possible to study deadenylation and decay kinetics and precursor and product relationships during mRNA decay.
The recognition of the importance of mRNA turnover in regulating eukaryotic gene expression has mandated the development of reliable, rigorous, and “user‐friendly” methods to accurately measure changes in mRNA stability in mammalian cells. Frequently, mRNA stability is studied indirectly by analyzing the steady‐state level of mRNA in the cytoplasm; in this case, changes in mRNA abundance are assumed to reflect only mRNA degradation, an assumption that is not always correct. Although direct measurements of mRNA decay rate can be performed with kinetic labeling techniques and transcriptional inhibitors, these techniques often introduce significant changes in cell physiology. Furthermore, many critical mechanistic issues as to deadenylation kinetics, decay intermediates, and precursor‐product relationships cannot be readily addressed by these methods. In light of these concerns, we have previously reported transcriptional pulsing methods based on the c‐fos serum‐inducible promoter and the tetracycline‐regulated (Tet‐off) promoter systems to better explain mechanisms of mRNA turnover in mammalian cells. In this chapter, we describe and discuss in detail different protocols that use these two transcriptional pulsing methods. The information described here also provides guidelines to help develop optimal protocols for studying mammalian mRNA turnover in different cell types under a wide range of physiologic conditions.
Chapter 22 Sensitive Detection of mRNA Decay Products by Use of Reverse-Ligation-Mediated PCR (RL-PCR)
2008, Methods in Enzymology
Ligation‐mediated PCR allows the detection and mapping of cleavage products of specific nucleic acid molecules out of complex nucleic acid mixtures. It can be applied to the detection of either degradation products of exogenously added nucleases in footprinting applications or natural decay products or reaction intermediates. We have developed various ligation‐mediated PCR approaches to analyze mRNAs, all relying on RNA ligation, followed by reverse‐transcription and PCR amplification. We have termed these approaches reverse‐ligation–mediated PCR (RL‐PCR). The ligation event involves either an RNA linker added to the 5′‐end of cleaved RNA or RNA circularization, allowing, respectively, the mapping and quantification of the cleavage points or the simultaneous analysis of the presence or absence of the 5′‐cap structure and the length of the poly(A) tail. These methods enabled us to develop a very efficient 5′‐RACE procedure to map mRNA 5′‐ends, to footprint in permeabilized cells the interaction of regulatory proteins with RNA, to detect the products of cellular ribozyme action and to analyze cellular decay pathways that involve deadenylation and/or decapping. I review herein the methodologic aspects and protocols of the various RL‐PCR procedures we have developed.

View all citing articles on Scopus

^☆: Lefkovits, I.

¹: To whom correspondence should be addressed. Fax: (713) 500–0652. E-mail:[email protected].

View full text

Regular ArticleTranscriptional Pulsing Approaches for Analysis of mRNA Turnover in Mammalian Cells☆

Abstract

Cell

Anal. Biochem.

Cell

Exp. Cell Res.

Anal. Biochem.

Cell

J. Biol. Chem.

Cell

J. Biol. Chem.

Annu. Rev. Biochem.

Microbiol. Rev.

Trends Biochem. Sci.

Control of Messenger RNA Stability

Genes Dev.

Mol. Cell. Biol.

Genes Dev.

The Immunology Methods Manual

Mol. Cell. Biol.

Regular Article
Transcriptional Pulsing Approaches for Analysis of mRNA Turnover in Mammalian Cells☆