Comparative dynamic transcriptome analysis (cDTA) reveals mutual feedback between mRNA synthesis and degradation

Abstract

To monitor eukaryotic mRNA metabolism, we developed comparative dynamic transcriptome analysis (cDTA). cDTA provides absolute rates of mRNA synthesis and decay in Saccharomyces cerevisiae (Sc) cells with the use of Schizosaccharomyces pombe (Sp) as an internal standard. cDTA uses nonperturbing metabolic labeling that supersedes conventional methods for mRNA turnover analysis. cDTA reveals that Sc and Sp transcripts that encode orthologous proteins have similar synthesis rates, whereas decay rates are fivefold lower in Sp, resulting in similar mRNA concentrations despite the larger Sp cell volume. cDTA of Sc mutants reveals that a eukaryote can buffer mRNA levels. Impairing transcription with a point mutation in RNA polymerase (Pol) II causes decreased mRNA synthesis rates as expected, but also decreased decay rates. Impairing mRNA degradation by deleting deadenylase subunits of the Ccr4-Not complex causes decreased decay rates as expected, but also decreased synthesis rates. Extended kinetic modeling reveals mutual feedback between mRNA synthesis and degradation that may be achieved by a factor that inhibits synthesis and enhances degradation.

Figure 1.

Design of a cDTA experiment. The Sc cells are labeled by adding 4tU into the media, whereas Sp cells are labeled by adding 4sU. The cells are then counted. Sc cells from different experiments are always mixed with the same amount of labeled Sp cells from a single batch. Cells are then lysed, RNA is extracted, biotinylated, and labeled RNA separated. Microarrays containing probes against both Sc and Sp transcripts are then used to quantify both total and labeled RNA.

Figure 2.

Establishing the cDTA protocol. (A) Assessment of cross-hybridization. Scatterplot of log intensities of 10,928 Affymetrix probe sets. The values on the x- resp. y-axis are obtained as the mean of two pure Sc resp. Sp replicate samples that were hybridized to the arrays. Sc and Sp probe sets (heat colored and gray scaled, respectively) can be separated almost perfectly. A total of 23 out of 5771 Sc probe sets show intensities above a (log) background intensity threshold of 4.5 in the Sp sample, whereas eight out of 5028 Sp probe sets were above background in the Sc sample. These 31 probe sets are regarded as affected by cross-hybridization (green circles). Of these, 16 probe sets were excluded from analysis because all probes were affected by cross-hybridization (Methods). (B) Linear measurement range. Exemplary illustration showing that the relation of mRNA concentration (real amount) and mRNA intensity (fluorescent scanner readout) follows the Langmuir adsorption model (Hekstra et al. 2003; Held et al. 2003, 2006; Skvortsov et al. 2007). The green line indicates linearity. (Black line) Sigmoidal behavior, resulting from noise at low-hybridization levels and saturation effects at high-hybridization levels. (Gray stripe) Linear measurement range that we defined as an intensity range of 4.5–8 (natural log basis) based on noise signals below 4.5, for example, for probes that detect transcripts of genes that were knocked out and based on observed saturation effects above 8. (C) Calibration of Sc:Sp cell mixture ratio. The optimal cell mixture ratio has been chosen to maximize the number of probes for both Sc and Sp that fall into the linear measurement range (B). Sc and Sp cells were mixed in Sc:Sp ratios of 1:1, 3:1, and 10:1. The respective median mRNA level ratios are 0.3, 0.95, and 3.02. Log (RNA intensity) distributions of Sc (red) and Sp (gray) are shown. The median intensity level of Sp is approximately three times higher than that of Sc. As a consequence, a Sc:Sp cell mixture ratio of 3:1 was used. (D) Comparison of the three different cell mixtures of (C) in pairwise log–log scatter plots. All arrays are normalized to a common median of 4052 Sp probe sets (gray-scaled). A total of 4475 Sc probe sets (those in the linear measurement range) are shown in heat colors. The parallel offset of the Sc probe sets from the main diagonal measures the mRNA level differences of Sc in the three cell mixtures. The differences should be 3.3, 10, and 3 when we plot Sc:Sp ratios of 10:1 vs. 3:1, 10:1 vs. 1:1, and 3:1 vs. 1:1, respectively. The corresponding measured offsets are 3.14, 9.46, and 3.01, and thus in very good agreement.

Figure 3.

cDTA normalization reveals global changes. (A) Determination of c_Sp, the ratio of labeled over total Sp mRNA. To obtain absolute synthesis and decay rates for Sc and Sp, we derived ratios of labeled to total RNA c_Sc and c_Sp for Sc and Sp, respectively. The c_Sc ratio was obtained in our previous study (Miller et al. 2011). To determine c_Sp, L_sc and T_sc are set to c_Sc and 1, respectively. L_sp and T_sp are then linearly rescaled. The resulting L_sp/T_sp is defined as c_Sp and then used in the further experiments as the global cDTA normalization factor. (B) cDTA normalization uses Sp signals as an internal standard. The bars indicate the median intensities of the array probe sets. Due to our experimental design, the ratio of labeled to total Sp RNA (c_Sp = L_sp/T_sp) must be the same in all experiments. To correct for differences in cell lysis, RNA extraction efficiency, and RNA purification efficiencies, the levels of Sp total and labeled mRNA are rescaled to the same values in all experiments. The Sc RNA levels are then corrected by median centering of Sp RNA levels. This normalization allows for a direct comparison of Sc data between experiments. Shown are both replicates for each of the four cDTA experiments.

Figure 4.

Comparison of cDTA with conventional methods. (A) Box plots of the expression distributions of the total and the labeled (newly synthesized) mRNA after cDTA normalization, obtained from the wild-type and the rpb1-1 mutant before, and 24 and 66 min after the shift to restrictive temperature. Transcriptional activity is roughly restored in both strains after 66 min. The global shifts in labeled expression are only slightly more pronounced in the rpb1-1 mutant, indicating a dominant role of heat shock in the profiles of rpb1-1. (B) Correlation analysis of mRNA half-life measurements. The heatmap shows pairwise Spearman correlation coefficients of half-life measurements (white: negative or zero correlation; purple: perfect correlation). The published half-life estimates except for Munchel et al. (2011) were obtained by experiments using transcriptional arrest. The estimates of Holstege et al. (1998), Wang et al. (2002), Grigull et al. (2004), and Shalem et al. (2008) were obtained using a yeast strain containing the Pol II temperature sensitive mutant rpb1-1. Dori-Bachash et al. (2011) used the transcription inhibitor 1,10-phenanthroline.

Figure 5.

Comparison of mRNA metabolism in Sp and Sc. (A) Scatter plot comparing mRNA decay rate folds versus synthesis rate folds of Sp and Sc transcripts encoding protein orthologs (>25% amino acid sequence identity). The offset of gray lines to parallel black lines indicates Sp:Sc ratios of median decay rates, synthesis rates, or total mRNA (0.20/0.83/2.72). Dashed gray lines indicate 1.5-fold changes from the median (gray lines). Color scheme corresponds to folds in total mRNA (magenta, positive log fold; green, negative log fold). A set of genes that show higher decay and synthesis rates (1.5-fold and adjusted P-value 0.5%), but almost unchanged (<1.5-fold) total mRNA (93 transcripts, striped area) was selected and tested with a Bayesian network-based gene-set analysis (MGSA) (Bauer et al. 2010). In this gene set, the ribosomal protein genes were enriched (blue dots; ellipse shows the 75% region of highest density). (B) Plots show log₂ fold distributions of total mRNA (gray), synthesis rate (red), and decay rate (blue) of Sp versus Sc transcripts encoding orthologous proteins as a function of amino acid sequence identity (%). Transcripts encoding highly conserved proteins such as ribosomal proteins are located on the right. They show more rapid turnover (synthesis and decay) in Sp, resulting in similar mRNA levels. (Solid black lines) Median log₂ fold; (shaded bands) central 80% regions. (Solid/dashed gray lines) Median log₂ fold of all orthologs/all genes.

Figure 6.

cDTA reveals changes in mRNA metabolism upon genetic perturbation. (A) Linear scatter plots (heat-colored) of mRNA synthesis rates, decay rates, and total mRNA levels in wild-type and mutant rpb1-N488D yeast strains as measured by cDTA. Slopes indicate global shift ratios of median synthesis rates, decay rates, and total mRNA of the rpb1-N488D mutant strain compared with wild type (0.26/0.31/0.75). (B) Alternative representation of the data from A in a single scatter plot comparing the changes in mRNA synthesis rates (log folds, x-axis) and decay rates (log folds, y-axis) in the rpb1-N488D mutant strain compared with the wild-type strain. Each point corresponds to one mRNA. The density of points is encoded by their brightness (grayscale). Contour lines define regions of equal density. The center of the distribution is located at (−1.8, −1.6), indicating that there is a global shift in the median synthesis rate by a factor of 0.26 (shift of the horizontal red line relative to the dashed x-axis line), and a global shift in the median decay rate by a factor of 0.31 (shift of the vertical red line relative to the dashed y-axis line). The global change in total mRNA levels is predicted by the offset of the diagonal red line from the dashed main diagonal, which corresponds to a change by a factor of 0.75. The number in brackets following this number (0.75) is the global change as it has been observed in the total mRNA measurements, which agrees well with the predicted number. The changes in total RNA levels do not exactly equal the quotient of synthesis and decay rate changes, due to an additional parameter for cell growth. (C) Scatter plots as in B comparing synthesis rates, decay rates, and total mRNA levels of Δccr4 and Δpop2 mutant strains to wild-type yeast. Ratios of median synthesis rates, decay rates, and total mRNA of the Δccr4/Δpop2 mutant strain compared with wild type are 0.49/0.39, 0.43/0.16, and 1.15/1.74, respectively. (D) Coupling of synthesis and decay rates, on the absolute level. For each condition, the median synthesis rate (y-axis) and degradation rate (x-axis) is shown (dark dots). (Dashed lines) Fold induction/repression relative to wild type. The dots lie approximately on a line with positive slope, indicating synthesis-decay compensation. A variation analysis for the estimation of the median synthesis and decay rates with cDTA has been performed. The ellipses show the 95% bootstrap confidence regions in each condition. The main axes of the ellipses reveal that the errors in the estimation of synthesis and decay rates are not independent, yet small enough to prove that the coupling is not due to estimation variance.