High guanine and cytosine content increases mRNA levels in mammalian cells

Abstract

Mammalian genes are highly heterogeneous with respect to their nucleotide composition, but the functional consequences of this heterogeneity are not clear. In the previous studies, weak positive or negative correlations have been found between the silent-site guanine and cytosine (GC) content and expression of mammalian genes. However, previous studies disregarded differences in the genomic context of genes, which could potentially obscure any correlation between GC content and expression. In the present work, we directly compared the expression of GC-rich and GC-poor genes placed in the context of identical promoters and UTR sequences. We performed transient and stable transfections of mammalian cells with GC-rich and GC-poor versions of Hsp70, green fluorescent protein, and IL2 genes. The GC-rich genes were expressed several-fold to over a 100-fold more efficiently than their GC-poor counterparts. This effect was not due to different translation rates of GC-rich and GC-poor mRNA. On the contrary, the efficient expression of GC-rich genes resulted from their increased steady-state mRNA levels. mRNA degradation rates were not correlated with GC content, suggesting that efficient transcription or mRNA processing is responsible for the high expression of GC-rich genes. We conclude that silent-site GC content correlates with gene expression efficiency in mammalian cells.

Figure 1. Distribution of Human Genes with Respect to Their GC3 Contents, and GC3 Contents of Genes Used in This Study

Data adapted from the Codon Usage Database [ 71].

Figure 2. Expression of Hsp70 (GC3 = 92%) and Hsc70 (GC3 = 46%)

(A) Three independent clones of pcDNA3-Hsp70-HA (GC3 = 92%) and six clones of pcDNA3-Hsc70-HA (GC3 = 46%) were used to transfect HeLa cells. 24 h following transfection the cells were harvested and the Hsc70-HA or Hsp70-HA protein levels were analyzed by Western blotting using an anti-HA antibody. An anti-GAPDH antibody was used as loading control. (B) Equal amounts of Hsp70 and Hsc70 mRNA were used as templates for in vitro translation in rabbit reticulocyte lysates in the presence of ³⁵S-Methionine. The reaction was initiated by the addition of reticulocyte lysate to the translation mix and samples were removed in 2-min intervals into SDS sample buffer. The reaction products were analyzed by SDS-PAGE and autoradiography. (C–E) HeLa cells were transfected with equal amounts of pcDNA3-Hsp70-HA or pcDNA3-Hsc70-HA plasmids. After 24 h, total cellular RNA was isolated and analyzed by qRT-PCR. The graphs represent Hsp or Hsc70 (C), neo (D), and GAPDH (E) mRNA amounts. Hsp70, cells transfected with pcDNA3-Hsp70-HA; Hsc70, cells transfected with pcDNA3-Hsc70-HA; control, untransfected cells. The mRNA amounts were normalized to the amounts in the Hsp70-transfected cells. The error bars represent standard deviations from three to four independent transfections.

Figure 3. Expression of EGFP (GC3 = 96%) and GFP(GC3 = 35%) in Transiently Transfected Cells

(A–C) HeLa cells were transfected with pGFP-N2 or pEGFP-N2 plasmids. 24 h following transfection, cells were trypsinized and washed, and GFP and EGFP protein levels were analyzed by flow cytometry. (A) Control cells. (B) Cells transfected with pGFP-N2. (C) Cells transfected with pEGFP-N2. The horizontal axes represent green fluorescence. (D and E) Expression of GFP and EGFP mRNA. HeLa cells were transfected with pGFP-N2 or pEGFP-N2 plasmids. After 24 h, total cellular RNA was isolated and analyzed by qRT-PCR. The graphs represent GFP or EGFP (D) and neo (E) mRNA amounts. Control, untransfected cells. The results are representative of three experiments.

Figure 4. Expression of Human Interleukin 2 (IL2) Variants in Transiently Transfected Cells

(A and B) HeLa cells were transfected with plasmids encoding the IL2 variants. 24 h following transfection, cell culture media were used for protein quantification by ELISA, and adhering cells for mRNA measurements by real-time RT-PCR. (A) ELISA measurement of IL2 protein levels using serial dilutions of culture media. Black squares, enhanced IL2 (eIL2, GC3 = 100%); white squares, IL2-eIL2 hybrid (IL2-eIL2, GC3 = 70%); black triangles, wild-type IL2 (IL2, GC3 = 41%); white triangles, weakened IL2 (wIL2, GC3 = 7%). The result is representative of three experiments. (B) Real-time RT-PCR measurement of IL2 mRNA. IL2 mRNA levels were normalized to GAPDH mRNA. Error bars represent standard deviations from two to four independent transfections using different plasmid preparations. (C and D) same as (A and B) using Saos-2 cells.

Figure 5. Expression of GFP and IL2 Variants in Stably Transfected MCF-7 Cells

MCF-7 cells were stably transfected with expression plasmids containing GFP (GC3 = 35%), EGFP (GC3 = 96%), wIL2 (GC3 = 7%), IL2 (GC3 = 41%), IL2-eIL2 (GC3 = 70%), eIL2 (GC3 = 100%), or with an empty pcDNA3.1 plasmid. The expression plasmids contained CMV promoters and were integrated in random genomic locations. Protein and mRNA was quantified in three to five individual clones for each transgene. (A) Flow cytometry measurements of GFP and EGFP protein levels. (B) Real-time RT-PCR measurements of GFP and EGFP mRNA levels. (C) ELISA measurements of the IL2 protein levels. (D) real-time RT-PCR measurements of IL2 mRNA levels. GFP and IL2 mRNA levels were normalized to GAPDH mRNA. The controls represent pcDNA3.1-transfected cells. The vertical axis in each graph represents arbitrary units.

Figure 6. Expression of GFP and IL2 Variants in Stably Transfected Flp-In T-Rex-293 Cells

The GFP or IL2 variants under the control of tetracycline-inducible CMV promoters were integrated into the single FRT site of Flp-In T-Rex-293 cells. Protein and mRNA was quantified in three to five individual clones for each transgene. (A) Flow cytometry measurements of GFP and EGFP protein levels 24 h post-induction. (B) Real-time RT-PCR measurements of GFP and EGFP mRNA levels 12 h post-induction. (C) ELISA measurements of the IL2 protein levels 24 h post-induction. (D) Real-time RT-PCR measurements of IL2 mRNA levels 24 h post-induction. GFP and IL2 mRNA levels were normalized to GAPDH mRNA. The controls represent CAT- transfected cells (A) or the parental Flp-In T-Rex-293 cell line (B and D). The vertical axis in each graph represents arbitrary units.

Figure 7. Stability of GC-Rich and GC-Poor mRNA in Mammalian Cells

HeLa cells were transfected with the indicated plasmids and after 20 h they were treated with 10 μg/mL actinomycin D. At the indicated times, mRNA was isolated and quantified by real-time RT-PCR. The GAPDH and c-myc mRNA levels represent the means of their levels in cells transfected with GC-rich and GC-poor genes. In each graph, little circles represent GAPDH and crosses, c-myc. (A) black circles, Hsp70; white circles, Hsc70. (B) black squares, EGFP; white squares, GFP. (C) Black triangles, eIL2; white triangles, IL2. The data is representative of two independent experiments.