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Tunable protein synthesis by transcript isoforms in human cells.

Floor SN, Doudna JA - Elife (2016)

Bottom Line: However, the relationship between human transcript diversity and protein production is complex as each isoform can be translated differently.We fractionated a polysome profile and reconstructed transcript isoforms from each fraction, which we term Transcript Isoforms in Polysomes sequencing (TrIP-seq).Select 5' untranslated regions exert robust translational control between cell lines, while 3' untranslated regions can confer cell type-specific expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States.

ABSTRACT
Eukaryotic genes generate multiple RNA transcript isoforms though alternative transcription, splicing, and polyadenylation. However, the relationship between human transcript diversity and protein production is complex as each isoform can be translated differently. We fractionated a polysome profile and reconstructed transcript isoforms from each fraction, which we term Transcript Isoforms in Polysomes sequencing (TrIP-seq). Analysis of these data revealed regulatory features that control ribosome occupancy and translational output of each transcript isoform. We extracted a panel of 5' and 3' untranslated regions that control protein production from an unrelated gene in cells over a 100-fold range. Select 5' untranslated regions exert robust translational control between cell lines, while 3' untranslated regions can confer cell type-specific expression. These results expose the large dynamic range of transcript-isoform-specific translational control, identify isoform-specific sequences that control protein output in human cells, and demonstrate that transcript isoform diversity must be considered when relating RNA and protein levels.

No MeSH data available.


Changes in protein production conferred by 5′ UTRs are more robust across cell lines than those conferred by 3′ UTRs.Plotted is the coefficient of variation (the standard deviation divided by the mean) for the indicated UTR fused to luciferase across five cell lines and HEK 293T cells at 2 hr and 18 hr (Figure 6).DOI:http://dx.doi.org/10.7554/eLife.10921.020
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fig6s1: Changes in protein production conferred by 5′ UTRs are more robust across cell lines than those conferred by 3′ UTRs.Plotted is the coefficient of variation (the standard deviation divided by the mean) for the indicated UTR fused to luciferase across five cell lines and HEK 293T cells at 2 hr and 18 hr (Figure 6).DOI:http://dx.doi.org/10.7554/eLife.10921.020

Mentions: Remarkably, the luciferase production from all tested 5′ leader reporters was qualitatively similar in all six conditions (Figure 6A). However, the difference between the two RICTOR 5′ leader sequences is decreased. In contrast, the 3′ UTR reporters show considerably more variability (Figure 6B). Calculation of the coefficient of variation of all reporters across all six conditions highlights the greater variability found in 3′ UTR reporters (Figure 6—figure supplement 1A). We additionally compared HEK 293T cells at 2-hr and 8-hr post-transfection to ascertain differences in RNA stability. Most 5′ leader reporters are similar between these two timepoints (Figure 6A), but 3′ UTR reporters show larger changes. Specifically, NAB1-001 and RICTOR-001 generate much more luciferase at the 2-hr timepoint, suggesting the long UTRs of these genes may promote RNA degradation leading to decreased protein at eighteen hours.10.7554/eLife.10921.019Figure 6.Translational control by transcript 5′ leader sequences is robust across cell types.


Tunable protein synthesis by transcript isoforms in human cells.

Floor SN, Doudna JA - Elife (2016)

Changes in protein production conferred by 5′ UTRs are more robust across cell lines than those conferred by 3′ UTRs.Plotted is the coefficient of variation (the standard deviation divided by the mean) for the indicated UTR fused to luciferase across five cell lines and HEK 293T cells at 2 hr and 18 hr (Figure 6).DOI:http://dx.doi.org/10.7554/eLife.10921.020
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4764583&req=5

fig6s1: Changes in protein production conferred by 5′ UTRs are more robust across cell lines than those conferred by 3′ UTRs.Plotted is the coefficient of variation (the standard deviation divided by the mean) for the indicated UTR fused to luciferase across five cell lines and HEK 293T cells at 2 hr and 18 hr (Figure 6).DOI:http://dx.doi.org/10.7554/eLife.10921.020
Mentions: Remarkably, the luciferase production from all tested 5′ leader reporters was qualitatively similar in all six conditions (Figure 6A). However, the difference between the two RICTOR 5′ leader sequences is decreased. In contrast, the 3′ UTR reporters show considerably more variability (Figure 6B). Calculation of the coefficient of variation of all reporters across all six conditions highlights the greater variability found in 3′ UTR reporters (Figure 6—figure supplement 1A). We additionally compared HEK 293T cells at 2-hr and 8-hr post-transfection to ascertain differences in RNA stability. Most 5′ leader reporters are similar between these two timepoints (Figure 6A), but 3′ UTR reporters show larger changes. Specifically, NAB1-001 and RICTOR-001 generate much more luciferase at the 2-hr timepoint, suggesting the long UTRs of these genes may promote RNA degradation leading to decreased protein at eighteen hours.10.7554/eLife.10921.019Figure 6.Translational control by transcript 5′ leader sequences is robust across cell types.

Bottom Line: However, the relationship between human transcript diversity and protein production is complex as each isoform can be translated differently.We fractionated a polysome profile and reconstructed transcript isoforms from each fraction, which we term Transcript Isoforms in Polysomes sequencing (TrIP-seq).Select 5' untranslated regions exert robust translational control between cell lines, while 3' untranslated regions can confer cell type-specific expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States.

ABSTRACT
Eukaryotic genes generate multiple RNA transcript isoforms though alternative transcription, splicing, and polyadenylation. However, the relationship between human transcript diversity and protein production is complex as each isoform can be translated differently. We fractionated a polysome profile and reconstructed transcript isoforms from each fraction, which we term Transcript Isoforms in Polysomes sequencing (TrIP-seq). Analysis of these data revealed regulatory features that control ribosome occupancy and translational output of each transcript isoform. We extracted a panel of 5' and 3' untranslated regions that control protein production from an unrelated gene in cells over a 100-fold range. Select 5' untranslated regions exert robust translational control between cell lines, while 3' untranslated regions can confer cell type-specific expression. These results expose the large dynamic range of transcript-isoform-specific translational control, identify isoform-specific sequences that control protein output in human cells, and demonstrate that transcript isoform diversity must be considered when relating RNA and protein levels.

No MeSH data available.