mRNA half-live determination

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2002; Shalem, 2008). Since the median mRNA half lives vary considerably throughout the different species, Yang and colleagues postulated the dependence between median transcript half live and cell cycle time (Yang, et al. 2003).

1.1.5 Post-transcriptional regulation of response to osmotic stress

Stability modulation of selected mRNAs has been observed to be one level that is used by MAP kinases to coordinate reorganisation of gene expression in response to environmental changes (Shalem, et al. 2008; Lai, et al. 2002; Molin, et al. 2009; Romero-Santacreu, et al. 2009; Grigull, et al. 2004). The human Hog1 homologue, p38, stabilizes the cytokine mRNAs regulating the binding of destabilization factor TTP (tristetraprolin) to AU-rich elements (ARE) in the 3’-UTR (Sandler, et al. 2008). The yeast mRNA tif51a, whose stability is regulated by its ARE, is destabilized when Hog1 function was inhibited (Vasudevan & Peltz, 2001). In S. cerevisiae, the hxt1 (encoding for transmembrane glucose transporter) mRNA was stabilized under osmotic stress conditions (Greatrix, et al. 2006).

Genome wide studies on mRNA stability in yeast suggested that mRNA decay contribute to genetic regulation of stress response and nutrient deprivation (Grigull et al. 2004). During the initial phase, global transcript stability decreases within 6 min after stress induction, whereas stress-responsive transcripts exhibit an increase in stability (Molin, et al. 2009; Romero-Santacreu, et al. 2009). After 30 min, a sharp decrease in mean stability of all initially stabilized stress related transcripts was observed, whereas stress-repressed genes become stabilized, indicating a cellular adaption to stress. The changes in stability between 6 and 30 min correlate with changes in steady-state levels between 30 and 60 min indicating that changes in transcript stability precede steady-state levels after osmotic shock. Hog1 affects both, steady-state levels and stability of stress-responsive transcripts and the modulation is dependent on the applied osmotic pressure (Molin, et al. 2009; Romero-Santacreu, et al. 2009): After treatment with 0.7 M

NaCl, the levels of induced mRNAs peak after 45 min (Rep, et al. 2000), while lower salt concentration causes earlier peaking (Posas, et al. 2000).

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rates uses labeled mRNA. While these principles have been successfully used for mammalian, insect and plant cells, this approach is not applicable for yeast.

1.2.1 Inhibitors

The first approach to measure mRNA stability in terms of half-lives and underlying kinetics is achieved by blocking transcription followed by analysis of mRNA abundance at several time points. Under the assumption that mRNA decay is a stochastic process and can be described by an exponential function, the change in mRNA abundance at any given time point is considered as first order process. Therefore, mRNA decay will be described by first derivation dC/dt, where C represents mRNA abundance present at time t. Assuming an idealized situation, in which transcription is completely inhibited at time-point t=0, the subsequent reduction of mRNA abundance during time-points t>0 is then a direct indication of mRNA half-live. To ensure a complete blockage of transcription at t=0, several Pol-II inhibitors have been used for studies in yeast and cultured cell lines of higher eukaryotes (for review see Ross, et al. 1995). As an overview, the predominantly used inhibitors and the temperature-sensitive Pol II mutant Rpb1-1 are described in the following paragraphs. Recent studies, however, revealed that these experimental approaches have some negative side effects on transcriptomics.

Actinomycin-D (ActD)

Several studies on mRNA half-live in yeast as well as in higher eukaryotes made use of Actinomycin-D (ActD), thiolutin and 1,10-Phenantrolin (Phen). For example, Raghavan, et al.

(2002) applied ActD to human T lymphocytes and identified short-lived mRNAs encoding for cytokines, cell surface receptors, signal transduction regulators, transcription factors, cell cycle regulators and regulators of apoptosis. ActD was recently used in archaea to analyze mRNA stability to identify novel RNA degrading characteristics (Evguenieva-Hackenbert, et al. 2008).

Narsai, et al. (2007) treated cultured arabidopsis thaliana cells with ActD and showed, that genes possessing at least one intron produce significantly more stable transcripts as intron-less genes. However, recent studies revealed some severe drawbacks of ActD. ActD affects the cellular ATP pool and produces therefore significant side effects by influencing other ATP-dependent processes (Ross, et al. 1995). This leads to considerable differences between results obtained with ActD and other methods utilizing less toxic compounds. Harrold, et al. (1991) compared values of mRNA half-lives of immunoglobulin heavy- and light chain encoding transcripts in mouse myeloma cells obtained from different methods. Surprisingly, the values ranged from 2.4 h (ActD) to 5.9 h (DRB). Additionally, actD binds to GC-rich sequences of DNA and inhibits RNA Pol II, which leads to DNA damage response and apoptosis. The DNA damage response activates several RNPs, such as human HuR, and AU-binding factor 1 (Auf1), which stabilize target mRNAs or modulate translation.

- 53 - 1,10-phenanthroline (Phen)

1,10-phenanthroline (Phen) works as a chelating agent that forms stable complexes by coordination of bivalent ions, especially with Ni²⁺, Zn²⁺ and Mg²⁺(Chang, et al. 1970; Chang, et al.

1978; Santiago, et al. 1986; Johnston, et al. 1994). The inhibitory effect on RNA polymerases has been described by Scrutton, et al. for E. coli (1971). Additionally, Phen has been observed to intercalate DNA, which probably contributes to shut-off transcription (Drew, et al. 1984). Phen has been deployed as inhibitor in a variety of studies on mRNA synthesis and halflive. Yin, et al.

(2000) used Phen to analyze changes in mRNA stability encoding for fbp1 and pck1 in response at low glucose levels. They could show, that low glucose levels strongly repress transcription of both, fbp1 and pck1, and additionally lead to accelerated degradation of the corresponding mRNAs. In a genome-wide study, Grigull, et al. (2004) compared the changes in mRNA levels between Phen, cordyceptin, actD and thiolution with the temperature-sensitive mutant Rpb1-1 in yeast. Among these inhibitors, Phen showed almost identical expression profile to Rpb1-1.

Inhibition of transcription by Phen exhibits some drawbacks: First, Phen works as metalchelator that probably sequesters Mg²⁺ in the active center of RNA polymerases and it is most likely, that other magnesium dependent processes or enzymes are affected by Mg²⁺ depletion. Second, Phen induces heat-shock response in yeast. After treatment with Phen, hsp82 mRNA reach same levels as in response to heat-shock (Adams, et al. 1991).

Pol-II temperature-sensitive mutant: Rpb1-1

The temperature-sensitive mutant Rpb1-1 has been identified by Nonet, et al. (1987). By shifting the temperature from 24⁰C to 36⁰C, the authors observed a detectable reduction in mRNA abundance after 15 min and after 45 min, a significant loss in global mRNA abundance (Nonet, et al. 1987). The availability of a ts-mutant which shuts off exclusively Pol-II transcription enabled a number of studies producing interesting results in mRNA turnover and promoted yeast to become a modelorganism for studies on mRNA decay (Herrick, et al. 1990;

Moore, et al. 1991; Li, et al. 1999; Grigull, et al. 2004; Wang, et al. 2002; Shalem, et al. 2008). For example, Holstege, et al. (1998) used the Rpb1-1 strain and combined transcriptional shut off with microarray analysis of global mRNA half-lives. With this approach, half-lives of 5735 mRNAs were calculated. However, a major drawback of this strategy lies in the temperature shift which is necessary to shutoff transcription completely. Several studies observed a high induction of heat-shock response genes which might introduce a stress dependent change in mRNA stability and therefore to a potential bias in mRNA half-live determination (Preiss, et al.

2003).

1.2.2 Genomic run on (GRO)

Genomic-run-on (GRO) has been developed to measure transcription rates and quantify mRNA abundance to obtain genomewide mRNA synthesis and decayrates under steady-state conditions (Birse, et al. 1997; Hirayoshi & Lis, 1999; Garcia-Martinez, et al. 2004). GRO is performed in three steps: First, S. cerevisiae cells are permeabilized in a cold sarkosyl buffer for 20 min which stops all physiological processes and disrupts all chromatin associated proteins with the exception of elongating RNA polymerases. Second, the run-on reaction is performed for 5 min in the presence of radioactive ³³P-UTP label, which is incorporated into nascent mRNA molecules during elongation. Third, transcripts are isolated and hybridized on custom made nylon-microarrays. The values obtained in the GRO-experiment are proportional to the average density of Pol II under the assumption of a constant Pol II elongation rate (Garcia-Martinez, et

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al. 2004). However, GRO is an opportunity to measure mRNA kinetics, but there are some limitations: First, GRO requires sarkosyl to permeabilize the cell wall to ensure uptake of the radioactive label. This causes an instantaneous loss of nucleosidetriphosphate and a complete chromatin disruption (Pérez-Ortin, 2008; Hirayoshi & Lis, 1999). Second, GRO measures nascent mRNA synthesis rates. Nascent mRNA undergoes several quality control and processing steps until the mature mRNA is prepared for translation.