RPL39L is an example of a recently evolved ribosomal protein paralog that shows highly specific tissue expression patterns and is upregulated in ESCs and HCC tumors

Abstract

Ribosomal proteins (RPs) have been shown to be able to impart selectivity on the translating ribosome implicating them in gene expression control. Many ribosomal proteins are highly conserved and recently a number of ribosomal protein paralogs have been described in mammals. We examined the expression pattern of RPs in differentiating mouse Embryonic Stem Cells (ESCs), paying particular attention to the RP paralogs. We find the RP paralog Rpl39l is highly expressed in ESC and its expression strongly correlates with hepatocellular carcinoma tumor (HCC) samples with high tumor grading and alpha-fetoprotein level giving it diagnostic potential. We further screen the expression pattern of all RPs and their paralogs across 22 different tissues. We find that the more recently evolved RP paralogs show a much greater level of tissue-specific expression. We propose that these RP paralogs evolved more recently to provide a greater level of gene expression control to higher eukaryotes.

Keywords: RPL39L; hepatocellular carcinoma; ribosomal protein; ribosomal protein paralogs.

Figure 1. Ribosomal protein gene expression in mouse and human Embryonic Stem Cells (ESCs). (A) Relative levels of 98 RP mRNAs detected in mouse ESCs and NPCs by RNA-sequencing. In total, 10 RPs exhibited ≥ 2-fold change in expression on ESC differentiation. The values are presented as fold change in ESCs relative to NPCs. (B) qRT-PCR validation of Rpl39l and Rpl10l in mouse ESCs and cells differentiated to NPCs. (C) qRT-PCR analysis of Rpl39l and Rpl10l in H9 human ESC and its derived fibroblasts.

Figure 2. High Rpl39l mRNA expressions in human cancer cell lines and HCC tumor samples. (A) Rpl39l and Rpl39 mRNA expression levels were examined in two human epithelial cell lines and 10 cancer cell lines using qRT-PCR analysis. Data shown was normalized to Gapdh. (B) qRT-PCR analysis of Rpl39l in a panel of 52 HCC tumors and adjacent non-tumoral liver tissue. Expression levels were normalized to Gapdh. (C–F) Statistical correlative analyses were conducted between Rpl39l expression and clinico-pathological features of HCC patients. Boxplots show the second and third quartiles, and the whiskers show the maximum and minimum values.

Figure 3. qRT-PCR analysis of 89 ribosomal protein mRNAs in 22 human tissues and a cell line. (A) Hierarchical clustering of the qPCR-derived expression levels segregates the RP genes into two clusters, with low or high expression levels across tissues. Red depicts high expression levels, whereas green and black correspond to low and unchanged expression levels, respectively. The color bar on the top shows the gene expression range from +3 to -3 in log₂ scale. (B) A distinct cluster of 19 heterogeneously expressed RP genes. (C) Dynamic expressions of the RP genes across 22 human tissues and cell line were calculated in terms of expression variation of RP gene expression. As highlighted by the black bar, Rpl39l exhibits the greatest expression heterogeneity across different tissues and cells.

Figure 4. Expression variation and evolutionary stage of 89 RP mRNAs in human tissues. (A) Correlation between gene expression variations of ribosomal proteins in human tissues with their corresponding E-stage. Four RP paralogs with high expression variation were highlighted in blue. The positive correlation of expression variation and E-stage was indicated with a dotted trend line. (B) Phylogenetic tree based on sequence similarity for ribosomal protein orthologs across different species. H, Homo sapiens; A, Arabidopsis thaliana; DR, Drosophila melanogaster; DA, Danio rerio; DI, Dictyostelium discoideum; and Y, yeast species. L or L1 in red indicates a ribosomal protein-like paralog.