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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.


Read Tracking Across the Sequencing Analysis Pipeline."Not RMSK" refers to reads not mapping to repeatmasker sequences, and "not abundant" is reads not mapping to rRNA and the mitochondrial chromosome, etc (Materials and methods).DOI:http://dx.doi.org/10.7554/eLife.10921.008
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fig1s3: Read Tracking Across the Sequencing Analysis Pipeline."Not RMSK" refers to reads not mapping to repeatmasker sequences, and "not abundant" is reads not mapping to rRNA and the mitochondrial chromosome, etc (Materials and methods).DOI:http://dx.doi.org/10.7554/eLife.10921.008

Mentions: To extract global trends in isoform-specific translation, we hierarchically clustered transcript isoform polysome profiles and selected eight clusters that are representative of general trends in the data (Figure 2; Materials and methods). The depth of sequencing (Figure 1—figure supplement 3), augmented by the fractionation strategy, enables detection of 62,703 transcript isoforms in the polysome profile (Figure 2—source data 1). Isoforms in the observed clusters exhibit diverse average patterns across polysomes (Figure 2A,B), from clusters 1 and 2, which contain isoforms primarily in high polysomes, to cluster 3 with isoforms in the middle, to clusters 6 and 7 where isoforms are in low polysomes. Independent clustering of the two biological replicate TrIP-seq datasets shows that the high- and low-polysome clusters (1, 2 and 6) appear more robust when comparing between averaged and individual replicate clusterings (Figure 2—figure supplement 1A). Many clusters have similar total polysome abundance but different distributions, indicating that to obtain accurate measurements of isoform-specific translatability it is crucial to fractionate the polysome profile.10.7554/eLife.10921.009Figure 2.Clustering of transcript distributions yields eight major clusters with diverse behavior across the polysome profile.


Tunable protein synthesis by transcript isoforms in human cells.

Floor SN, Doudna JA - Elife (2016)

Read Tracking Across the Sequencing Analysis Pipeline."Not RMSK" refers to reads not mapping to repeatmasker sequences, and "not abundant" is reads not mapping to rRNA and the mitochondrial chromosome, etc (Materials and methods).DOI:http://dx.doi.org/10.7554/eLife.10921.008
© Copyright Policy
Related In: Results  -  Collection

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

fig1s3: Read Tracking Across the Sequencing Analysis Pipeline."Not RMSK" refers to reads not mapping to repeatmasker sequences, and "not abundant" is reads not mapping to rRNA and the mitochondrial chromosome, etc (Materials and methods).DOI:http://dx.doi.org/10.7554/eLife.10921.008
Mentions: To extract global trends in isoform-specific translation, we hierarchically clustered transcript isoform polysome profiles and selected eight clusters that are representative of general trends in the data (Figure 2; Materials and methods). The depth of sequencing (Figure 1—figure supplement 3), augmented by the fractionation strategy, enables detection of 62,703 transcript isoforms in the polysome profile (Figure 2—source data 1). Isoforms in the observed clusters exhibit diverse average patterns across polysomes (Figure 2A,B), from clusters 1 and 2, which contain isoforms primarily in high polysomes, to cluster 3 with isoforms in the middle, to clusters 6 and 7 where isoforms are in low polysomes. Independent clustering of the two biological replicate TrIP-seq datasets shows that the high- and low-polysome clusters (1, 2 and 6) appear more robust when comparing between averaged and individual replicate clusterings (Figure 2—figure supplement 1A). Many clusters have similar total polysome abundance but different distributions, indicating that to obtain accurate measurements of isoform-specific translatability it is crucial to fractionate the polysome profile.10.7554/eLife.10921.009Figure 2.Clustering of transcript distributions yields eight major clusters with diverse behavior across the polysome profile.

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.