Well hell.. if anyone is interested and bored.. here's what I've written so far:
2. Gene expression may be controlled at various levels, including transcription, mRNA stabilization, or translation. How would you establish which mechanism or mechanisms may be operative?
Techniques such as ELISA or densitometry are useful methods to evaluate the response of cells to a given stimulus. However, these approaches offer very little insight to the investigator as to which molecular mechanism is engaged by the cell to evoke a differential response. It then becomes necessary to incorporate more discriminatory techniques into the study design so as to elucidate the mechanism by which a particular cellular response is elicited.
Several techniques are available for the detection of differential gene expression at the level of steady-state mRNA. These include Northern Blot, RT-PCR, slot-blot hybridization, microarray analysis, and others. By measuring the production of gene specific mRNA, an investigator can gain information on the potential for modulation of protein production. However, while variance in transcript level often correlates with changes in protein expression, it is critical to remember that changes in mRNA levels do not guarantee differential expression of protein. These data require validation by looking at actual protein expression through the use of other techniques.
Regulation of protein expression also occurs at the post-transcriptional level. These mechanisms of control include regulation of nuclear export, cytoplasmic localization, translation initiation, or mRNA decay (8). To examine if nuclear export or cytoplasmic localization where an operative mechanism in differential protein expression, in situ hybridization could be used to identify in detail how mRNA is being trafficked within the cell (9). In particular, Long, et al. have developed a technique capable of monitoring both the temporal, as well as the spatial, trafficking of mRNA using fluorescent in situ hybridization (10).
It is also important to distinguish between increased rates of transcription versus mRNA stability when trying to determine the molecular mechanism by which cells respond to a particular stimulus. Fan, et al., working with human non-small lung carcinoma H1299 cells subject to stress, demonstrated that only half of observed changes in mRNA levels within their model were accompanied by a corresponding increase or decrease in transcription rate (11). Transcription rate was measured using the nuclear run-on assay, a technique in which cell nuclei are isolated, incubated with radio labeled nucleotides, and hybridized to probe arrays to determine which genes are being actively transcribed at a given time point. Cheadle, et al. modified the nuclear run-on assay for high throughput use compatible with microarray analysis. Studying changes in human Jurkat T cells undergoing activation, their group discovered that about half of the 2,386 regulated genes studied underwent modulation of mRNA levels with no corresponding change in transcription rate, implying biochemical changes had occurred within the cell effecting mRNA stability and rate of decay (12). These studies emphasize the importance of mRNA stability and rate of transcript decay as a critical mechanism by which regulation of gene expression is achieved.
Previously, adenine and uridine rich elements (ARE) in the 3' untranslated region of cytokine mRNA have been identified which contribute to transcript metabolism (13, 14). An array of ARE-binding proteins are capable of modulating both mRNA stability, as well as translation, in a positive or negative fashion (15). For instance, it has been shown that when DLD-1 cells are exposed to a cytokine mixture containing IFN-γ, IL-1β, and TNF-α, the mRNA stabilizer HuR was able to compete for and displace the destabilizing KH-type splicing regulatory protein from a common binding site, leading to a diminished decay of inducible nitric oxide synthase (iNOS) mRNA (16). It was later reported that iNOS is upregulated in MSC treated with a similar cocktail, and secretion of this factor is important in their immunosuppressive function (17). Our group has shown IFN-γ treated MSC to possess heightened efficacy in vivo (18), and in lieu of these other studies, regulation of mRNA metabolism is a potential effector mechanism of activated MSC which would warrant further investigation.
Mammalian transcript half-lives range from ~15 minutes to as much as 10 hours (19), and may span several cell cycles (20). Modulation of this mRNA metabolism is a powerful mechanism by which a cell is able to manipulate its expression profile, and an alternate approach to nuclear run-on is needed to prove if changes in mRNA stability occur in response to a particular treatment. Such a technique would require the ability to quantify specific transcript levels across time. One such approach would be to generate a library of mRNA primers that contain radio labeled nucleotides. Knowing the specific activity of the radio labels would allow the investigator to quantify the levels of a specific transcript over time, and by comparing these data to control experiments would demonstrate if changes in mRNA stability were to occur. Alternatively, transcriptional inhibitors (actinomycin-D, thiolutin, 1,10-phenanthroline, 6-azauricil, cordycepin, etc.) or a temperature-sensitive RNA polymerase II, as demonstrated by Grigull, et al, could be used to block de novo transcription, allowing one to quantify transcript levels over time using microarray analysis (19).
An additional mechanism of control by which protein expression may be regulated is at the level of translation. Kaur, et al. have provided evidence that Akt activation is required for IFN-stimulated engagement of the mTor/p70 S6 kinase pathway (7). The mTor/p70 S6 kinase pathway regulates translation through the downstream effectors ribosomal protein S6 and eukaryotic initiation factor 4E (21). In these studies, initiation of translation was verified by isolating polysomal mRNA before qRT-PCR using a sucrose density gradient. A similar technique could be used to purify stress granules, which are cytoplasmic foci that inhibit translation, disassemble polysomes, and contain RNA-induced silencing complexes (RISC) (22).
In summary, there are a number of different molecular mechanisms by which cells are capable of regulating gene expression for the production of differential responses. It is important for a more in-depth understanding of how a system works to keep these mechanisms in mind when developing an experimental design or attempting to critically evaluate data. Only through discrete consideration of each of these mechanisms can an investigator channel the biological potential of their system for the most effective development of potential therapies or scientific application.
