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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.


Extended validation of TrIP-seq isoform abundances across polysome fractions using qRT-PCR.(A) qRT-PCR (top) and TrIP-seq abundances (bottom) for three EEF1B2 isoforms. (B) As in (A) except for the gene SRSF5. qRT-PCR abundance is calculated as a fold change versus cytoplasmic abundance. Note that systematic normalization differences exist between the TrIP-seq and qRT-PCR abundances since the TrIP-seq abundances are from repeat and rRNA subtracted data, while the input for qRT-PCR is total RNA. NTC: no template control.DOI:http://dx.doi.org/10.7554/eLife.10921.007
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fig1s2: Extended validation of TrIP-seq isoform abundances across polysome fractions using qRT-PCR.(A) qRT-PCR (top) and TrIP-seq abundances (bottom) for three EEF1B2 isoforms. (B) As in (A) except for the gene SRSF5. qRT-PCR abundance is calculated as a fold change versus cytoplasmic abundance. Note that systematic normalization differences exist between the TrIP-seq and qRT-PCR abundances since the TrIP-seq abundances are from repeat and rRNA subtracted data, while the input for qRT-PCR is total RNA. NTC: no template control.DOI:http://dx.doi.org/10.7554/eLife.10921.007

Mentions: To confirm the sequencing data, we first analyzed beta-actin (ACTB), which is known to be heavily translated with many ribosomes on each message, and migrates with high polysomes in a gradient (Figure 1C; (Sterne-Weiler et al., 2013; Zhang et al., 2015). However, ACTB only has one transcript isoform, so we examined ATF4, which has one dominant isoform exhibiting low-polysome association in TrIP-seq data and by RT-PCR (Figure 1C), consistent with it being translationally repressed in the absence of cellular stress (Harding et al., 2000). We then analyzed eukaryotic elongation factor 1 beta 2 (EEF1B2), which has three isoforms with the same coding sequence but alternative 5′ leaders as a representative gene that exhibits isoform-specific ribosome association (Figure 1—figure supplement 1B). The isoform-specific polysome profiles and RT-PCR conducted from polysome fractions agree qualitatively, validating the accuracy of the isoform-level quantifications (Figure 1D). Weaker amplification of the longer isoform (EEF1B2-003) in high polysomes may be due to PCR bias towards smaller amplicons (Walsh et al., 1992), and replicate experiments show EEF1B2-003 present in high polysomes (Figure 1—figure supplement 1C). We additionally validated the TrIP-seq data using qRT-PCR from polysome fractions and find that the two measurements agree for both EEF1B2 and SRSF5 (Figure 1—figure supplement 2). Both coding mRNAs and long noncoding RNAs (lncRNAs) associate with ribosomes (Ingolia et al., 2014; van Heesch et al., 2014), so we measured their polysomal abundance. Protein-coding genes and isoforms are found across the polysome, but noncoding and lncRNA genes are predominantly in the low polysome fractions indicating that they are generally weakly ribosome associated or in other large macromolecular complexes (Figure 1E and Figure 1—figure supplement 1D).


Tunable protein synthesis by transcript isoforms in human cells.

Floor SN, Doudna JA - Elife (2016)

Extended validation of TrIP-seq isoform abundances across polysome fractions using qRT-PCR.(A) qRT-PCR (top) and TrIP-seq abundances (bottom) for three EEF1B2 isoforms. (B) As in (A) except for the gene SRSF5. qRT-PCR abundance is calculated as a fold change versus cytoplasmic abundance. Note that systematic normalization differences exist between the TrIP-seq and qRT-PCR abundances since the TrIP-seq abundances are from repeat and rRNA subtracted data, while the input for qRT-PCR is total RNA. NTC: no template control.DOI:http://dx.doi.org/10.7554/eLife.10921.007
© Copyright Policy
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC4764583&req=5

fig1s2: Extended validation of TrIP-seq isoform abundances across polysome fractions using qRT-PCR.(A) qRT-PCR (top) and TrIP-seq abundances (bottom) for three EEF1B2 isoforms. (B) As in (A) except for the gene SRSF5. qRT-PCR abundance is calculated as a fold change versus cytoplasmic abundance. Note that systematic normalization differences exist between the TrIP-seq and qRT-PCR abundances since the TrIP-seq abundances are from repeat and rRNA subtracted data, while the input for qRT-PCR is total RNA. NTC: no template control.DOI:http://dx.doi.org/10.7554/eLife.10921.007
Mentions: To confirm the sequencing data, we first analyzed beta-actin (ACTB), which is known to be heavily translated with many ribosomes on each message, and migrates with high polysomes in a gradient (Figure 1C; (Sterne-Weiler et al., 2013; Zhang et al., 2015). However, ACTB only has one transcript isoform, so we examined ATF4, which has one dominant isoform exhibiting low-polysome association in TrIP-seq data and by RT-PCR (Figure 1C), consistent with it being translationally repressed in the absence of cellular stress (Harding et al., 2000). We then analyzed eukaryotic elongation factor 1 beta 2 (EEF1B2), which has three isoforms with the same coding sequence but alternative 5′ leaders as a representative gene that exhibits isoform-specific ribosome association (Figure 1—figure supplement 1B). The isoform-specific polysome profiles and RT-PCR conducted from polysome fractions agree qualitatively, validating the accuracy of the isoform-level quantifications (Figure 1D). Weaker amplification of the longer isoform (EEF1B2-003) in high polysomes may be due to PCR bias towards smaller amplicons (Walsh et al., 1992), and replicate experiments show EEF1B2-003 present in high polysomes (Figure 1—figure supplement 1C). We additionally validated the TrIP-seq data using qRT-PCR from polysome fractions and find that the two measurements agree for both EEF1B2 and SRSF5 (Figure 1—figure supplement 2). Both coding mRNAs and long noncoding RNAs (lncRNAs) associate with ribosomes (Ingolia et al., 2014; van Heesch et al., 2014), so we measured their polysomal abundance. Protein-coding genes and isoforms are found across the polysome, but noncoding and lncRNA genes are predominantly in the low polysome fractions indicating that they are generally weakly ribosome associated or in other large macromolecular complexes (Figure 1E and Figure 1—figure supplement 1D).

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.