Genomic Insights into the Different Layers of Gene Regulation in Yeast
Figure 2
Outline of the different genomic techniques for TR and mRNA stability determination used in yeast. Determination of nascent TR in the nucleus is based on the detection of RNA polymerases or nascent RNAs. (a) Native chromatin can be purified and nascent RNA can be isolated. Then it is converted into cDNA and used for the tiling array analysis [32]. Alternatively, cells can be used for the GRO analysis (see Figure 1). (b) Cells can be fixed with formaldehyde and subjected to chromatin fragmentation and immunoprecipitation with RNA pol II antibodies. From this ternary complex, either DNA or RNA can be detached from polymerases and analyzed downstream. Recovered DNA is suitable for array hybridization (ChIP-Chip) [10, 33, 34] or HTS (ChIP-Seq) [35], whereas RNA can be converted into DNA and subjected to HTS [36]. The appearance of recently synthesized mRNA (newborn, orange) in the cytoplasm can be followed by thiouridine in vivo labeling, which is purified, quantified, and compared with the non labeled old mRNA (green). With this technique, it is possible to calculate mature TR and mRNA half-lives [37]. mRNA half-lives can also be calculated from a transcription shutoff experiment [38], from TR and RA data by assuming the steady state ( = TR/RA), or even for non-steady-state conditions [39]. DR: degradation rate. Other symbols are as in Figure 1.
