The decay of mRNA plays an important role in the regulation of gene expression. Although relatively ignored for many years and regarded as a simple ribonucleotide salvage pathway, mRNA decay has been established in recent years as a well-defined cellular process that plays an integral role in determining gene expression. The recent application of microarray methods to the study of diverse organisms will help us to better understand these gene regulatory circuits and the influence of transcript stability on gene expression. DNA microarray technology is the method of choice to study individual mRNA half-lives on a global scale. It is important to standardize these methods to generate reproducible and reliable results. In this chapter, we describe experimental designs for the analysis of mRNA decay on a genome-wide scale and provide detailed protocols for each experimental step. We also present an analysis of the decay of chromosomally encoded mRNAs in E. coli.

RNase E, an essential endoribonuclease of Escherichia coli , interacts through its C-terminal region with multiple other proteins to form a complex termed the RNA degradosome. To investigate the degradosome's proposed role as an RNA decay machine, we used DNA microarrays to globally assess alterations in the steady-state abundance and decay of 4,289 E. coli mRNAs at single-gene resolution in bacteria carrying mutations in the degradosome constituents RNase E, polynucleotide phosphorylase, RhlB helicase, and enolase. Our results show that the functions of all four of these proteins are necessary for normal mRNA turnover. We identified specific transcripts and functionally distinguishable transcript classes whose half-life and abundance were affected congruently by multiple degradosome proteins, affected differentially by mutations in degradosome constituents, or not detectably altered by degradosome mutations. Our results, which argue that decay of some E. coli mRNAs in vivo depends on the action of assembled degradosomes, whereas others are acted on by degradosome proteins functioning independently of the complex, imply the existence of structural features or biochemical factors that target specific classes of mRNAs for decay by degradosomes.

Highly parallel hybridization of nucleic acids on glass slides has successfully been applied to measure RNA and DNA abundances in Escherichia coli (1–4). In this chapter, we summarize our experience in working with E. coli DNA microarrays accumulated over a 4-yr period. Typically, we printed and used E. coli DNA microarrays containing roughly 6000 spotted elements. These included 4200 amplicons of E. coli open reading frames (ORFs), 112 amplicons of genes encoding stable RNAs, and more than 1500 control elements and replicates. We describe the methods for total RNA extraction, mRNA enrichment of total RNA, cDNA labeling via direct and indirect incorporation of fluorophors, and microarray hybridization. Additionally, we present strategies for optimizing microarray hybridizations and descriptions of several Internet-based tools useful in analyzing data from array experiments.

Recently, several groups have used high-density DNA microarrays to study mRNA turnover. These new data suggest that decay contributes significantly to determining mRNA levels, and they should prompt us to refocus our attention on the regulatory potential of mRNA decay.

The Escherichia coli endoribonucleases RNase E (Rne) and RNase G (Rng) have sequence similarity and broadly similar sequence specificity. Whereas the absence of Rne normally is lethal, we show here that E. coli bacteria that lack the rne gene can be made viable by overexpression of Rng. Rng-complemented cells accumulated precursors of 5S ribosomal RNA (rRNA) and the RNA component of RNase P (i.e. M1 RNA), indicating that normal processing of these Rne-cleaved RNAs was not restored by RNase G; additionally, neither 5S rRNA nor M1 RNA was generated from precursors by RNase G cleavage in vitro. Using DNA microarrays containing 4405 Escherichia coli open reading frames (ORFs), we identified mRNAs whose steady-state level was affected by Rne, Rng or the N-terminal catalytic domain of RNase E. Most transcript species affected by RNase E deficiency were also elevated in an rne deletion mutant complemented by Rng. However, approximately 100 mRNAs that accumulated in Rne-deficient cells were decreased by rng-complemention, thus identifying targets whose processing or degradation may be the basis for RNase E essentiality. Remarkably prominent in this group were mRNAs implicated in energy-generating pathways or in the synthesis or degradation of macromolecules.

Much of the information available about factors that affect mRNA decay in Escherichia coli, and by inference in other bacteria, has been gleaned from study of less than 25 of the ≈4,300 predicted E. coli messages. To investigate these factors more broadly, we examined the half-lives and steady-state abundance of known and predicted E. coli mRNAs at single-gene resolution by using two-color fluorescent DNA microarrays. An rRNA-based strategy for normalization of microarray data was developed to permit quantitation of mRNA decay after transcriptional arrest by rifampicin. We found that globally, mRNA half-lives were similar in nutrient-rich media and defined media in which the generation time was approximately tripled. A wide range of stabilities was observed for individual mRNAs of E. coli, although ≈80% of all mRNAs had half-lives between 3 and 8 min. Genes having biologically related metabolic functions were commonly observed to have similar stabilities. Whereas the half-lives of a limited number of mRNAs correlated positively with their abundance, we found that overall, increased mRNA stability is not predictive of increased abundance. Neither the density of putative sites of cleavage by RNase E, which is believed to initiate mRNA decay in E. coli, nor the free energy of folding of 5′ or 3′ untranslated region sequences was predictive of mRNA half-life. Our results identify previously unsuspected features of mRNA decay at a global level and also indicate that generalizations about decay derived from the study of individual gene transcripts may have limited applicability.