Stochastic mRNA synthesis in mammalian cells

Abstract

Individual cells in genetically homogeneous populations have been found to express different numbers of molecules of specific proteins. We investigated the origins of these variations in mammalian cells by counting individual molecules of mRNA produced from a reporter gene that was stably integrated into the cell's genome. We found that there are massive variations in the number of mRNA molecules present in each cell. These variations occur because mRNAs are synthesized in short but intense bursts of transcription beginning when the gene transitions from an inactive to an active state and ending when they transition back to the inactive state. We show that these transitions are intrinsically random and not due to global, extrinsic factors such as the levels of transcriptional activators. Moreover, the gene activation causes burst-like expression of all genes within a wider genomic locus. We further found that bursts are also exhibited in the synthesis of natural genes. The bursts of mRNA expression can be buffered at the protein level by slow protein degradation rates. A stochastic model of gene activation and inactivation was developed to explain the statistical properties of the bursts. The model showed that increasing the level of transcription factors increases the average size of the bursts rather than their frequency. These results demonstrate that gene expression in mammalian cells is subject to large, intrinsically random fluctuations and raise questions about how cells are able to function in the face of such noise.

Figure 1. Detection and Quantification of Single Molecules of mRNA in Individual Cells

(A) Schematic diagram depicting the mRNA detection method. Multiple fluorescent probes bind to each mRNA molecule, yielding a bright, localized signal. (B) Merged image of a three-dimensional stack of images from a CHO cell expressing the 7x-tetO gene depicted in (A), where each mRNA is hybridized to FISH probes that bind to the multimeric probe-binding sequence in its 3′-UTR (probe P1-TMR binding to the M1 multimer). Each spot corresponds to a single mRNA molecule. (C) Identification of the spots in the three-dimensional image stack in (C). Each particle found by the image-analysis algorithm is colored differently, showing that the algorithm is accurate and that individual molecules are uniquely identified. The scale bars are 5 μm long.

Figure 2. Cell-to-Cell Variation of mRNA Numbers in Clonal Cell Lines

(A) Schematic diagram of the doxycycline-controllable promoters and the reporter genes that they control. Doxycycline binds to the tTA protein, thereby preventing it from binding to the tet operator. (B, C) Representative fields of cells from cell lines E-YFP-M1-1x and E-YFP-M1-7x, containing the 1x-tetO and 7x-tetO promoters, respectively, where each mRNA is hybridized to FISH probe P1-TMR and the image was obtained by merging a three-dimensional stack of images. (D) Two sister cells from cell line E-YFP-M1-7x displaying mRNA hybridized to FISH probe P1-TMR (red) and costained with DAPI (blue). The image represents one focal plane. The scale bars are 5 μm long.

Figure 3. Statistical Analysis of Per-Cell mRNA Population Distributions

(A) Histograms showing the distribution of mRNA molecules per cell for three doxycycline concentrations for both cell lines E-YFP-M1-1x (left) and E-YFP-M1-7x (right). (B) Graphs showing the population mean (top) and noise (defined as the standard deviation divided by the mean [bottom]) as a function of doxycycline concentration. Statistics were taken from the mRNA counts used in (A), and error bars were obtained by bootstrapping. (C) Activation rate (λ/δ) and average number of mRNA molecules produced per burst (μ/γ) for 1x-tetO (blue) and 7x-tetO (red) obtained from fitting the mRNA count data to the model of gene activation and inactivation by the maximum-likelihood method. Error bars reflect 95% confidence intervals.

Figure 4. Cell-to-Cell Variations in mRNA Numbers in a Cell Line with Multiple Reporter Gene Integrations at the Same Gene Locus

(A) Representative field from cell line L-GFP-M1-7x, generated by lipofection, where the mRNA was hybridized to FISH probe P1-TMR; the image was obtained by merging a three-dimensional stack of images. (B) Histogram showing the distribution of mRNA molecules per cell over for cell line L-GFP-M1-7x when grown in media containing no doxycycline. (C) Graphs showing the population mean (top) and noise (defined as the standard deviation divided by the mean [bottom]) as a function of doxycycline concentration. Error bars were obtained by bootstrapping.

Figure 5. Dual-Reporter Experiments Showing Variations Are Intrinsically Random and Can Affect an Entire Gene Locus

(A) Schematic depicting dual-reporter integration experiments, where the two reporters are either integrated in separate locations in the genome (left) or at the same locus (right). (B) Two-color overlay showing a merged three-dimensional stack of images of the cell line in which two different reporter genes (one expressing mRNA containing the M1 sequence array and the other expressing mRNA containing the M2 sequence array) are integrated into separate loci (cell line L-GFP-M1-7x+L-CFP-M2-7x); green corresponds to the signal from GFP-M1 mRNA and red corresponds to the signal from CFP-M2 mRNA (using probes P1-TMR and P2-Alexa-594, respectively). The relative mRNA levels of both genes were quantified by counting the mRNA of each color in a single optical slice in each cell (inset). (C) Two-color overlay showing a merged three-dimensional stack of images of the cell line in which the two distinct reporter genes are integrated into the same locus (cell line L-YFP-M1-CFP-M2), where the same FISH probes were used as in (B). The relative mRNA levels of both genes were quantified by counting the mRNA of each color in a single optical slice in each cell (inset). The scale bars are 5 μm long.

Figure 6. Cell-to-Cell Variations in the mRNA Encoding the Large Subunit of RNA Polymerase II

(A) Representative field of cell line E-YFP-M1-7x upon performing FISH with differently colored probes for both YFP-M1 mRNA and the mRNA encoding the large subunit of RNA polymerase II. The image is a two-color overlay, where green corresponds to the signal from YFP-M1 mRNA (one optical slice) and red corresponds to the signal from RNA polymerase mRNA (merged three-dimensional image stack). The probes used were P1-Cy5.5 and P3-TMR. (B) Histogram showing the distribution of RNA polymerase mRNA molecules per cell (top) and scatterplot (bottom) showing reporter mRNA levels (quantified by counting the mRNA in a single optical slice) and RNA polymerase mRNA levels (quantified by counting all mRNA) in each cell. Gene activation (λ/δ), inactivation (γ/δ), and transcription rates (μ/δ) (normalized by the mRNA decay rate δ) are given with 95% confidence intervals as indicated. The scale bar is 5 μm long.

Figure 7. Propagation of mRNA Variations to Variations in Protein Levels

(A) Scatterplot of total GFP and mRNA numbers in individual cells from cell line L-GFP-M1-7x grown under conditions of no doxycycline (blue), 0.08 ng/ml doxycycline (red), and 0.16 ng/ml doxycycline (green). Marginal histograms indicate the distribution of reporter mRNA per cell (top) or total GFP (right) for all the growth conditions. (B) Scatterplot of total CFP and mRNA levels (obtained from a single optical slice) in individual cells from cell line L-GFP-M1-7x+L-CFP-M2-7x grown under conditions of no doxycycline. Marginal histograms indicate the distribution of reporter mRNA per cell (top) or total GFP (right) for all the growth conditions. (C) Histogram of total YFP per cell from cell line E-YFP-M1-7x. YFP was quantified in live cells to minimize loss of fluorescence due to fixation and permeabilization. (D) Scatterplots and associated marginal histograms showing the results of stochastic simulations of the model of mRNA and protein dynamics presented in Protocol S1. The mRNA dynamics were the same for all cases, but the protein degradation rate was increased as indicated. Details of the simulation procedures and exact values of the parameters used are given in the Materials and Methods section.