GC Content of Early Metazoan Genes and Its Impact on Gene Expression Levels in Mammalian Cell Lines

Abstract

With the genomes available for many animal clades, including the early-branching metazoans, one can readily study the functional conservation of genes across a diversity of animal lineages. Ectopic expression of an animal protein in, for instance, a mammalian cell line is a generally used strategy in structure-function analysis. However, this might turn out to be problematic in case of distantly related species. Here we analyzed the GC content of the coding sequences of basal animals and show its impact on gene expression levels in human cell lines, and, importantly, how this expression efficiency can be improved. Optimization of the GC3 content in the coding sequences of cadherin, alpha-catenin, and paracaspase of Trichoplax adhaerens dramatically increased the expression of these basal animal genes in human cell lines.

Fig. 1.

—The GC% and GC(3) in genomes and coding sequences (CDS) across metazoan and nonmetazoan species. (Left) Cladogram showing the evolutionary relationships among the organisms used in this study. (Right) The percentages of the GC content of the whole genome, of the CDS, and the third-codon position GC-content (GC3) of the CDS.

Fig. 2.

—The distribution of GC and GC3 content of the protein coding sequences in vertebrate, insect and nonbilaterian species. GC (light blue) and GC3 (dark blue) density plots of the CDS of vertebrates (A) Homo sapiens (human) and (B) Danio rerio (zebrafish), of (C) Drosophila melanogaster (fruit fly), and of non-bilaterians (D) Trichoplax adhaerens (placozoan), (E) Nematostella vectensis (cnidarian), and (F) Amphimedon queenslandica (porphyrian).

Fig. 3.

— Expression of human (Hs), N. vectensis (Nv), and T. adhaerens (Ta) cDNAs in HeLa and HEK293T cells. Equal amounts of pdcDNAMyc-(Hs/Nv/Ta)-Cdh1-Cyto, pdcDNAMyc-(Hs/Nv/Ta)-Ctnna2-Nterm and pdcDNAFlag-Ta-Pcasp plasmids were used to transfect HeLa or HEK293T cells. (A, C, E) Protein levels were analyzed by Western blotting using an anti-Myc antibody or an anti-Flag antibody as indicated; an anti-vinculin antibody was used as a control. (B, D, F) Expression of (Hs/Nv/Ta)-Cdh1-Cyto, (Hs/Nv/Ta)-Ctnna2-Nterm and Ta-Pcasp mRNA. After 24 h, total cellular RNA was isolated and analyzed by qRT-PCR. As negative controls, untransfected HeLa or HEK293T cells were used as well as transfection with empty pdcDNAMyc plasmid (mock). Values have been normalized such that the values of the different WT constructs of T. adhaerens are equal to 1. The results are representative of three experiments.

Fig. 4.

—Correlation between GC content and gene expression efficiency in T. adhaerens. Protein abundance factors per animal for 6,500 proteins (Ringrose et al. 2013) are plotted against the GC content of the corresponding genes. The yellow line represents the trend line for the abundance factors and the red line represents the trend line for the GC content.