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. 2012 Dec;18(12):2299-305.
doi: 10.1261/rna.035865.112. Epub 2012 Oct 25.

Alternative transcription start site selection leads to large differences in translation activity in yeast

Maria F Rojas-Duran  1 , Wendy V Gilbert
Affiliations

Alternative transcription start site selection leads to large differences in translation activity in yeast

Maria F Rojas-Duran et al. RNA. 2012 Dec.

Abstract

mRNA levels do not accurately predict protein levels in eukaryotic cells. To investigate contributions of 5' untranslated regions (5' UTRs) to mRNA-specific differences in translation, we determined the 5' UTR boundaries of 96 yeast genes for which in vivo translational efficiency varied by 80-fold. A total of 25% of genes showed substantial 5' UTR heterogeneity. We compared the capacity of these genes' alternative 5' UTR isoforms for cap-dependent and cap-independent translation using quantitative in vitro and in vivo translation assays. Six out of nine genes showed mRNA isoform-specific translation activity differences of greater than threefold in at least one condition. For three genes, in vivo translation activities of alternative 5' UTR isoforms differed by more than 100-fold. These results show that changing genes' 5' UTR boundaries can produce large changes in protein output without changing the overall amount of mRNA. Because transcription start site (TSS) heterogeneity is common, we suggest that TSS choice is greatly under-appreciated as a quantitatively significant mechanism for regulating protein production.

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Figures

FIGURE 1.
FIGURE 1.
Some yeast genes have very heterogeneous 5′ UTRs. (A) Histogram of yeast genes' relative translation efficiencies (TE) as determined by Ingolia et al. (2009). TEs for the 96 genes selected for study are indicated below. (B) 5′ UTR lengths as determined by 5′ RACE for 96 genes. (C) Comparison of 5′ RACE results with 5′ UTR boundaries from published cDNAs. Each dot represents the median 5′ UTR length determined for one gene. A total of 27 out of 96 genes were represented by a single cDNA in Miura et al. (2006) and 32 genes were not represented. rs indicates the Spearman rank correlation of 5′ UTR lengths from 5′ RACE clones and cDNAs.
FIGURE 2.
FIGURE 2.
Alternative 5′ UTR isoforms differ in translation efficiency in vitro. (A) The longest and shortest 5′ UTR variants for nine genes with heterogeneous 5′ UTRs. (B) Schematic of reporter constructs and translation assay. (C) Representative ethidium bromide stained gels used to normalize luciferase activity to mRNA concentrations. (D) Mean normalized luciferase activity from translation of capped (5′-m7GpppG) mRNAs. Error bars represent standard deviations. P-values determined by Student's t-test (two-tailed) are indicated above the bars; (*) P < 0.05; (**) P < 0.01; (***) P < 0.001. (E) Comparison of mean translation activity to (left) 5′ UTR length and (right) predicted stability as determined by free-energy minimization using mfold (Zuker 2003).
FIGURE 3.
FIGURE 3.
Alternative 5′ UTR isoforms differ in translation efficiency in vivo. (A) Schematic of reporter constructs and translation assay. (B) Mean normalized luciferase activity from translation of alternative 5′ UTR mRNAs. Error bars represent standard deviations.
FIGURE 4.
FIGURE 4.
Alternative 5′ UTR isoforms differ in capacity for cap-independent translation. (A) Representative ethidium bromide stained gels used to normalize luciferase activity to mRNA concentrations. (B) Mean normalized luciferase activity from translation of uncapped (5′-GTP) mRNAs. Error bars represent standard deviations. P-values were calculated by Student's t-test (two-tailed); (*) P < 0.05; (**) P < 0.01; (***) P < 0.001. (C) Comparison of mean translation activity to 5′ UTR length and (D) predicted stability as determined by free-energy minimization using mfold (Zuker 2003).
FIGURE 5.
FIGURE 5.
Model for the role of transcription start-site selection in translational control of gene expression. (A) Turning genes on and off by switching between poorly translated and efficiently translated 5′ UTR isoforms. (B) Heterogeneous transcription start-site selection produces 5′ UTR isoforms that respond differently to global changes in translation factor activity. In this example, the “long” 5′ UTR variant is better translated than the corresponding “short” isoform under stress conditions, causing reduced cap-dependent initiation due to sequestration of eIF4E.
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