Agami and co-workers [1] concede that models and interpretations of global mRNA decay data can be easily oversimplified, and so new approaches will be needed to fully understand the dynamic properties of RNA regulation. Thus, one has to begin with simple assumptions and progressively refine the tools over time to address the complexity of this problem. Indeed, many issues surrounding experimental design and interpretation of mRNA stability data are not settled. The main one is the means by which transcription is experimentally inhibited, so that the data truly represent the balance between transcription and RNA stability without interference from chemical side-effects of the procedures. Conditional genetic switches are not available in mammalian cells for global analysis of mRNA decay, but temperature-sensitive mutants of RNA polymerase can be used with Saccharomyces cerevisiae, so that other cellular processes are minimally disturbed [6, 9].
An alternative method of distinguishing the contributions of transcription and RNA stability to mRNA abundance in mammalian cells is nuclear run-on followed by microarray analysis or other high-throughput RNA quantification tools [2, 10]. When applied to T-cell activation by Cheadle et al. [10] this approach revealed that at least 50% of the changes in accumulated mRNA levels detected by microarray were due to changes in RNA stability [10], and many workers believe that this is an underestimate. More recent use of thiolated nucleosides incorporated into nascent transcripts followed by capture of biotinylated transcripts has been used to quantify the transcriptional contribution to mRNA levels in response to cell activation before RNA processing can take place. Those changes in mRNA levels due to transcription can then be compared with RNA stability measurements of the population to evaluate the relative contributions of each process to the accumulated quantity of mRNA [8]. Interestingly, in the case of trypanosomes, one can avoid some of these technical concerns altogether while examining posttranscriptional regulation directly because transcription is not taking place during differentiation. Under these circumstances, co-regulated changes in mRNA levels that depend upon RNA stability alone can be quantified by microarray analysis. Several recent studies have demonstrated these coordinated changes of functionally related subsets of mRNAs during developmental stages of trypanosomes, consistent with the posttranscriptional operon/regulon model [5, 11]. Given the large number of identified RNA-binding proteins in the trypanosome genome, ribonucleoprotein immunoprecipitation followed by microarray (RIP-chip) and related methods should be able to identify the combinations of factors responsible for coordinating trypanosome posttranscriptional RNA regulon decay dynamics. Indeed, determining the combinatorial interactions of RNA-binding proteins and noncoding RNAs that coordinate the dynamic changes of mRNA subpopulations following cellular activation will provide insights into mechanisms of coordinated global gene expression [2, 4, 7].
As shown in these studies, efforts to understand the underlying principles of mRNA stability are essential because the balance between transcription and decay influences most, if not all, responses of cells to endogenous and exogenous signals. However, the ability to correlate changes in RNA stability with changes in protein output is tenuous given the poor correlation between the transcriptomic profiles and proteomic profiles. Moreover, given that each mRNA has multiple copies and that some copies probably reside in different states of localization, stability and translation, one cannot yet infer how quantification of any one state reflects on the other states. Thus, mRNA decay curves represent an average of the stability changes of all copies of each mRNA regardless of their individual states. It is logical to expect that the regulatory factors associated with each individual mRNA copy dictate in aggregate and coordinate these observed patterns of mRNA stability. It will be important to identify these combinatorially associated factors during cell-activation events and to quantify the dynamic interactions among them that govern cell growth and development.