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. 1999 Sep 14;96(19):10632-6.
doi: 10.1073/pnas.96.19.10632.

Messenger RNA translation state: the second dimension of high-throughput expression screening

Q Zong  1 , M Schummer, L Hood, D R Morris
Affiliations

Messenger RNA translation state: the second dimension of high-throughput expression screening

Q Zong et al. Proc Natl Acad Sci U S A. .

Abstract

Technological advances over the past 10 years have generated powerful tools for parallel analysis of complex biological problems. Among these new technologies, DNA arrays have provided an important experimental approach for identifying changes in the levels of individual mRNA molecules during important cellular transitions. However, cellular behavior is dictated not by mRNA levels, but by the proteins translated from the individual mRNA species. We report a high-throughput method for simultaneously monitoring the translation state and level of individual mRNA species. Messenger RNAs from resting and mitogenically activated fibroblasts were separated, according to degree of ribosome loading, into well-translated and under-translated pools. cDNA probes generated from these fractions were used to interrogate cDNA arrays. Among approximately 1,200 genes analyzed, less than 1% were found to be translationally regulated in response to mitogenic activation, demonstrating the strong selectivity of this regulatory mechanism. This high-throughput approach is shown to be an effective tool for superimposing translation profile on mRNA level for large numbers of genes, as well as for identifying translationally regulated genes for further study.

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Figures

Figure 1
Figure 1
Flow chart for high-throughput analysis of translationally controlled genes. A summary of mRNA distribution changes in human fibroblasts of all the array elements on CLONTECH Altas human Cancer Expression Array is given in the graph at the bottom of the figure. The change of mRNA translation state is defined by the following formula: formula image AT, AU, RT, and RU are defined in the text. A value greater than 1 indicates that the mRNA moved from the under-translated fraction into the translated fraction upon growth activation. If the ratio is less than 1, the value is inverted and a negative number is used to indicate a change in the reverse direction.
Figure 2
Figure 2
Translation state of human genes monitored on cDNA arrays. Comparison of DNA arrays hybridized with complex 32P-labeled cDNA probes from the under-translated (Left) and translated (Right) RNA pools of both resting (Lower) and serum-activated human foreskin fibroblasts (Upper). Growth-arrested human foreskin fibroblasts were stimulated to enter the cell cycle by addition of serum and fractionated by centrifugation through a 0.5–1.5 M sucrose gradient (9). The RNA fractions containing one ribosome or less per mRNA were pooled as the under-translated fraction, and fractions loaded with two or more ribosomes per mRNA were pooled as translated fractions (Fig. 1). An equal percentage of the RNA in each fraction was used to prepare complex 32P-labeled first-strand cDNA probes as recommended by CLONTECH. CLONTECH Altas human cancer cDNA Expression Array filters were hybridized with the 32P-labeled cDNA probes. The arrows indicate the translationally regulated genes found in this study; 1, vimentin; 2, Stat1; and 3, 23-kDa highly basic protein.
Figure 3
Figure 3
Comparing translation states (A) and relative levels (B) of mRNAs in growth-arrested and serum-activated human fibroblasts. The hybridization signal of each element on the human cancer cDNA expression array was quantified and normalized to the median value of all the elements on the filter. A subset of genes was selected for graphing according to the following selection criteria: (i) specific hybridization signals were found on all four human cancer expression array filters and (ii) hybridization signals were consistent among the duplicates of the particular gene. (A) The translation state of a particular gene was expressed as the ratio of the normalized hybridization signal intensities between the well-translated and under-translated RNA fractions. The change of mRNA translation state was defined in the legend to Fig. 1. The arrows indicate the genes that were identified as translationally regulated on the human DNA array and confirmed on the polysome display and Northern blot analysis in this study: 1, vimentin; 2, Stat1; and 3, 23-kDa highly basic protein. (B) The relative mRNA level was expressed as the ratio of the normalized total hybridization signal intensities between the serum-activated and growth-arrested cells. The total hybridization signal intensity was the sum of the signal on both translated and under-translated array filters.
Figure 4
Figure 4
Polysome association in resting and activated human foreskin fibroblasts of mRNAs encoding vimentin, 23-kDa highly basic protein and β-actin. Cells were growth-arrested by serum starvation and stimulated by addition of serum (18). Cytoplasmic extracts were prepared and separated on sucrose gradients (9). (Upper) Optical density profiles of sucrose gradient. (Lower) Northern blot analysis of the RNA extracted from sucrose gradient fractions.
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