Systematic analysis of cis-elements in unstable mRNAs demonstrates that CUGBP1 is a key regulator of mRNA decay in muscle cells

Abstract

Background: Dramatic changes in gene expression occur in response to extracellular stimuli and during differentiation. Although transcriptional effects are important, alterations in mRNA decay also play a major role in achieving rapid and massive changes in mRNA abundance. Moreover, just as transcription factor activity varies between different cell types, the factors influencing mRNA decay are also cell-type specific.

Principal findings: We have established the rates of decay for over 7000 transcripts expressed in mouse C2C12 myoblasts. We found that GU-rich (GRE) and AU-rich (ARE) elements are over-represented in the 3'UTRs of short-lived mRNAs and that these mRNAs tend to encode factors involved in cell cycle and transcription regulation. Stabilizing elements were also identified. By comparing mRNA decay rates in C2C12 cells with those previously measured for pluripotent and differentiating embryonic stem (ES) cells, we identified several groups of transcripts that exhibit cell-type specific decay rates. Further, whereas in C2C12 cells the impact of GREs on mRNA decay appears to be greater than that of AREs, AREs are more significant in ES cells, supporting the idea that cis elements make a cell-specific contribution to mRNA stability. GREs are recognized by CUGBP1, an RNA-binding protein and instability factor whose function is affected in several neuromuscular diseases. We therefore utilized RNA immunoprecipitation followed by microarray (RIP-Chip) to identify CUGBP1-associated transcripts. These mRNAs also showed dramatic enrichment of GREs in their 3'UTRs and encode proteins linked with cell cycle, and intracellular transport. Interestingly several CUGBP1 substrate mRNAs, including those encoding the myogenic transcription factors Myod1 and Myog, are also bound by the stabilizing factor HuR in C2C12 cells. Finally, we show that several CUGBP1-associated mRNAs containing 3'UTR GREs, including Myod1, are stabilized in cells depleted of CUGBP1, consistent with the role of CUGBP1 as a destabilizing factor.

Conclusions: Taken together, our results systematically establish cis-acting determinants of mRNA decay rates in C2C12 myoblast cells and demonstrate that CUGBP1 associates with GREs to regulate decay of a wide range of mRNAs including several that are critical for muscle development.

Figure 1. Analysis of mRNA decay rate in C2C12 cells.

(A) Examples of mRNA decay curves were derived by the nonlinear least squares method for a long and a short half life mRNA (see Materials and Methods for details). (B) Distribution of mRNA half lives (see Dataset S1 for the complete list). The 10^th-percentile and 90^th-percentile values (indicated by red dotted lines) were used to select mRNAs with short and long half lives, respectively. The median value (2.9 hr) is indicated by a red line.

Figure 2. Destabilizing and stabilizing elements in 3′UTRs have combinatorial effects on mRNA stability.

(A) Top 20 ranked hexamers significantly enriched in the 3′UTRs of mRNAs with short and long half-lives. P-values were derived from Fisher's exact test. (B) Destabilizing and stabilizing elements (DEs and SEs) were derived by grouping significant hexamers (see Figure S1), and presented as sequence logos. (C) DEs and SEs have different frequencies of occurrence in 3′UTRs of mRNAs with different half-lives. mRNAs were divided into 4 groups based on their half life (shown in Figure 1B), i.e. 0–10%, 10–50%, 50–90%, and 90–100%. For each element, the frequencies of occurrence were standardized across the 4 groups by calculating (x-mean)/sd, where x is frequency of occurrence for an element in a group, and mean and sd are mean and standard deviation of frequencies of occurrence for the element in all groups.

Figure 3. mRNA decay is influenced by different elements in different cell types.

(A) Scatter plots comparing mRNA half lives in C2C12 with those in pluripotent and differentiating embryonic stem (ES) cells . ES/LIF− and ES/RA+ represent ES cells differentiated by withdrawal of Leukemia Inhibitory Factor (LIF) and addition of Retinoic Acid (RA), respectively. Pearson correlation coefficient (r) and P-value for linear regression are shown for each plot. (B) Heat maps showing significance of hexamers associated with mRNAs with short (left panel) and long (right panel) half lives in C2C12, ES, ES/LIF−, and ES/RA+. Top 20 ranked hexamers for each cell type were selected and combined to illustrate variable significance in different cells. Each hexamer has a Significance Score (SS). SS = −log(P-value)*s, where P-value was derived from Fisher's exact test, and s = 1 if the hexamer is significantly associated with mRNAs with short half life, and  = −1 otherwise. SS are shown in the heat map with color according to the scale shown in the figure. Hexamers were clustered (Euclidean distance and average linkage) for easy visualization.

Figure 4. Identification of CUGBP1-associated mRNAs.

(A) Western blot showing efficient immunoprecipitation of CUGBP1 from cytoplasmic extracts of C2C12 (LKO1) cells (upper panel). RT-PCR assays showing specific association of several transcripts with CUGBP1 immunoprecipitates. Gapdh, cMyc and Plk2 are negative controls. (B) Schematic of ribonucleoprotein immunoprecipitation microarray (RIP-Chip) experiment. The signal-to-negative ratio is the mean of probe set values of immunoprecipitated samples (α-CUGBP1) to that of negative control samples (control IgG). (C) Distribution of signal-to-negative ratios (see Dataset S2 for the complete list). The 95^th-percentile value (indicated in the graph) was used as the cut-off for mRNAs showing a positive association with CUGBP1. The ratios for transcripts assayed by RT-PCR in (A) and Figure S3 are shown as dots with red and green colors representing positive and negative immunoprecipitation, respectively.

Figure 5. CUGBP1 bound mRNAs contain GREs in their 3′UTRs.

(A) Top 20 ranked significant hexamers in the 3′UTRs of mRNAs with signal-to-negative ratios above 95^th-percentile. P-values were derived from Fisher's exact test comparing frequency of occurrence in top 5% transcripts with that in other transcripts. Consecutive Us > = 3 are underlined, and UGU is shown in red. (B) Comparison of signal-to-negative ratios in the CUGBP1 RIP-Chip experiment with mRNA half lives in C2C12 cells. A gene density plot was used to show the relationship, in which mRNAs were evenly divided into 20 groups based on signal-to-negative ratio (x-axis) or half life (y-axis). The number of mRNAs in each cell of the 20×20 table (observed value, obs) was normalized to the mean of all cells (expected value). The ratios (observed/expected) are shown in a heat map according to the color scale shown in the figure. Red represents enrichment, and Blue for depletion.

Figure 6. CUGBP1 bound mRNAs are stabilized in CUGBP1 knockdown cells.

The decay rates of the indicated mRNAs were assessed in C2C12 (LKO1) and CUGBP1 KD cells by qRT-PCR following transcription inhibition with actinomycin D. Abundance of the mRNA of interest was normalized to Gapdh at each time point. Each half life was measured three times in each cell line. Representative results are depicted.