Translational profiling in childhood acute lymphoblastic leukemia: no evidence for glucocorticoid regulation of mRNA translation

Background Glucocorticoids (GCs) are natural stress induced steroid hormones causing cell cycle arrest and cell death in lymphoid tissues. Therefore they are the central component in the treatment of lymphoid malignancies, in particular childhood acute lymphoblastic leukemia (chALL). GCs act mainly via regulating gene transcription, which has been intensively studied by us and others. GC control of mRNA translation has also been reported but has never been assessed systematically. In this study we investigate the effect of GCs on mRNA translation on a genome-wide scale. Results Childhood T- (CCRF-CEM) and precursor B-ALL (NALM6) cells were exposed to GCs and subjected to “translational profiling”, a technique combining sucrose-gradient fractionation followed by Affymetrix Exon microarray analysis of mRNA from different fractions, to assess the translational efficiency of the expressed genes. Analysis of GC regulation in ribosome-bound fractions versus transcriptional regulation revealed no significant differences, i.e., GC did not entail a significant shift between ribosomal bound and unbound mRNAs. Conclusions In the present study we analyzed for the first time possible effects of GC on the translational efficiency of expressed genes in two chALL model systems employing whole genome polysome profiling. Our results did not reveal significant differences in translational efficiency of expressed genes thereby arguing against a potential widespread regulatory effect of GCs on translation at least in the investigated in vitro systems. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-14-844) contains supplementary material, which is available to authorized users.

presents a list of all biotypes on the microarray. Figure 2: Comparison of the genes' relative expressions in pool 3 in EtOH-treated control samples between C7H2 and NALM6 cells. Left: all genes detectable on the Exon microarray, middle: protein coding genes, and right: genes from all other biotypes. The colors of the data points represent the differential expression of a gene between the C7H2 and the NALM6 cell lines, with red and blue indicating higher expression in C7H2 and NALM6 cells, respectively, and white corresponding to same expression in both cell lines.. Table 1: Average expression and relative expression in the various pools of all genes detected on the Exon microarray in C7H2 cells. Columns "transcript id", "gene id", "probe count", "gene name", "gene biotype" and "chromosome name" contain annotations for the respective probe set id on the microarray. Columns "RE.p1", "RE.p2", "RE.p3" contain averaged relative expression across the 3 biological replicates of each gene in pools 1, 2 and 3 respec-tively. Columns "exprs.p1", "exprs.p2" and "exprs.p3" contain averaged expression of a gene across the 3 biological replicates (log2 scale). Suffix "GC" and "EtOH" indicate the treatment.

GO-analysis on genes with similar translational efficiency
Genes were sorted based on their translational efficiency and grouped into 5 similar-sized groups of genes. A Gene Ontology (Biological Function) analysis was performed on each of these groups using the complete set of genes as background gene set. Supplementary Table 3: Significantly enriched Biological Process GO-terms in both cell lines; analysis using genes with the highest translational efficiencies in each cell line. "Size": total number of genes annotated to the GO-term, "Size": number of genes from those tested for enrichment that are annotated to the GO-term. Columns with suffix "C" and "N" contain values for C7H2 and NALM6 cells, respectively.  Table 4: Significantly enriched Biological Process GO-terms in both cell lines; analysis using genes with the lowest translational efficiencies in each cell line. "Size": total number of genes annotated to the GO-term, "Size": number of genes from those tested for enrichment that are annotated to the GO-term. Columns with suffix "C" and "N" contain values for C7H2 and NALM6 cells, respectively.      Analysis of microRNA target genes miRNA (family) target gene predictions base on Targetscan version 6.2 [1] (target genes have been defined using the file Predicted Targets Info.txt that contains all predicted conserved targets of conserved miRNA families).

Enriched processes in C7H2 cells
In this analysis we evaluated whether genes with low translatoinal efficiciency harbor similar miRNA target sites. Similar to a GO analysis, we thus performed hypergeometric tests to analyse for significant over-representation of predicted miRNA target sites in the 3' UTR of the 5% or genes with the lowest translational efficieny.
Of the in total 536 genes constituting the 5% of genes with lowest translational efficiency in C7H2 341 are also predicted target of at least one miRNA. These genes have been analysed for enrichment against the background gene set consisting of 6899 genes also predicted to be target of at least one miRNA (out of in total 10318 expressed protein coding genes). For NALM6 cells 363 genes have been tested against 7192 background genes (of in total 577 and 11129 test and background genes, respectively).
An enrichment analysis has also been performed on the 5% of the genes with the highest translational efficiency. Some of the miRNA families below (indicated by ‡) have also been found significant in that analysis and thus most likely represent false positive findings. Supplementary Table 15: miRNA families with a significant over-representation of predicted target sites in the 3' UTR of the 5% of genes with the lowest translational efficiency. C7H2 cell line. Count: number of tested genes being a predicted target of the miRNA, Size: total number of predicted target genes for the miRNA detectable on the microarray, %: percentage of predicted target genes being among the genes with the lowest translational efficiency. †: highest expression of any of the miRNAs host genes detected in the respective cell line. For miRNA host gene prediction see [2]. ‡: target sites for that miRNA are also significantly over-represented in 3' UTRs of genes with the highest translational efficiencies. Supplementary Table 16: miRNA families with a significant over-representation of predicted target sites in the 3' UTR of the 5% of genes with the lowest translational efficiency. NALM6 cell line. Count: number of tested genes being a predicted target of the miRNA, Size: total number of predicted target genes for the miRNA detectable on the microarray, %: percentage of predicted target genes being among the genes with the lowest translational efficiency. †: highest expression of any of the miRNAs host genes detected in the respective cell line. ‡: target sites for that miRNA are also significantly over-represented in 3' UTRs of genes with the highest translational efficiencies.

Detailed information on some miRNA families
Here we list some more information for some of the miRNA families identified in the analysis above. In addition to the expression of their target genes across all 3 pools we add information about the expression of these miRNAs in the analysed cell lines or the lymphoid tissue. Specifically, we evaluate expression of the mature miRNA in C7H2 cells [2], tissue specific expression according to www.microrna.org and information from the literature. miR-30abcdef/30abe-5p/384-5p • Host gene: host gene relatively high expressed in C7H2 and NALM6 cells.
Supplementary Figure 3: Expression of target genes for the miRNA family miR-30abcdef/30abe-5p/384-5p across pools in both cell lines. The boxplot represents expression of all target genes for the miRNA, expression of target genes among the 5% of genes with the lowest translational efficiency are draw in red. The horizontal blue line indicates the (highest) expression of any of the potential host genes for that miRNA in the respective cell line.
Supplementary Figure 4: Expression of target genes for the miRNA family miR-93/93a/105/106a/291a-3p/294/295/302abcde/372/373/428/519a/520be/520acd-3p/1378/1420ac across pools in both cell lines. The boxplot represents expression of all target genes for the miRNA, expression of target genes among the 5% of genes with the lowest translational efficiency are draw in red. The horizontal blue line indicates the (highest) expression of any of the potential host genes for that miRNA in the respective cell line.
• Host gene: average expression of one of the host genes.
Supplementary Figure 5: Expression of target genes for the miRNA family miR-181abcd/4262 across pools in both cell lines. The boxplot represents expression of all target genes for the miRNA, expression of target genes among the 5% of genes with the lowest translational efficiency are draw in red. The horizontal blue line indicates the (highest) expression of any of the potential host genes for that miRNA in the respective cell line.
• Host gene: moderate expression of the host gene in NALM6.
• Expression profile from microrna.org: high in lymphoma.