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Increased functional protein expression using nucleotide sequence features enriched in highly expressed genes in zebrafish.

Horstick EJ, Jordan DC, Bergeron SA, Tabor KM, Serpe M, Feldman B, Burgess HA - Nucleic Acids Res. (2015)

Bottom Line: Zebrafish embryos are rapidly injected with calibrated doses of mRNA, enabling the effects of multiple sequence changes to be compared in vivo.These results suggested principles for increasing protein yield in zebrafish through biomolecular engineering.Rational gene design thus significantly boosts expression in zebrafish, and a similar approach will likely elevate expression in other animal models.

View Article: PubMed Central - PubMed

Affiliation: Program in Genomics of Differentiation, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, MD 20892, USA.

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Structural mRNA and gene features in highly expressed genes. (a) Box plots of the maximal free energy of folding (dG) for nucleotides from −4 to +37 for genes in each data set and for genes in the mouse and human Refseq databases. *P < 0.05 for the mean compared to the zebrafish Refseq set. (b) Cumulative frequency histograms for the free energy of the minimum energy secondary structure (dG) for Refseq (black) and Himix (blue) gene sets. Dotted line indicates that for 90% of highly expressed genes, the dG was greater than –13.1 kcal/mol. (c) Distribution of 3′UTRs lengths (from the stop codon to the beginning of the polyadenylated sequence) in the Refseq (gray) and Himix (blue) sets. Bins sizes are 100 bp with maximum values per bin indicated on the x-axis. Inset, mean and standard error for each group. Mann–Whitney U test * P < 0.001. (d) Distribution of number of introns per gene for the Refseq (gray) and Himix (blue) sets.
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Figure 3: Structural mRNA and gene features in highly expressed genes. (a) Box plots of the maximal free energy of folding (dG) for nucleotides from −4 to +37 for genes in each data set and for genes in the mouse and human Refseq databases. *P < 0.05 for the mean compared to the zebrafish Refseq set. (b) Cumulative frequency histograms for the free energy of the minimum energy secondary structure (dG) for Refseq (black) and Himix (blue) gene sets. Dotted line indicates that for 90% of highly expressed genes, the dG was greater than –13.1 kcal/mol. (c) Distribution of 3′UTRs lengths (from the stop codon to the beginning of the polyadenylated sequence) in the Refseq (gray) and Himix (blue) sets. Bins sizes are 100 bp with maximum values per bin indicated on the x-axis. Inset, mean and standard error for each group. Mann–Whitney U test * P < 0.001. (d) Distribution of number of introns per gene for the Refseq (gray) and Himix (blue) sets.

Mentions: Translation initiation is impaired when strong secondary structure for nucleotides −4 to +37 (relative to the AUG) reduces accessibility for the ribosome (11,61). In other parts of the transcript, local mRNA structure is not generally a major determinant of the rate of translation elongation since the ribosome processively destabilizes the mRNA (62,63). The mean free energy of the minimum energy structure (dG) at 28°C for zebrafish transcripts was 2 kcal/mol greater than for mouse or human transcripts, suggesting that nucleotide sequence has adapted to reduce secondary structure at the typical environmental temperatures that zebrafish inhabit (16.5–33°C (64); Figure 3a). Highly expressed genes showed a small but significant tendency for a more open mRNA structure than other genes (Figure 3a and b).


Increased functional protein expression using nucleotide sequence features enriched in highly expressed genes in zebrafish.

Horstick EJ, Jordan DC, Bergeron SA, Tabor KM, Serpe M, Feldman B, Burgess HA - Nucleic Acids Res. (2015)

Structural mRNA and gene features in highly expressed genes. (a) Box plots of the maximal free energy of folding (dG) for nucleotides from −4 to +37 for genes in each data set and for genes in the mouse and human Refseq databases. *P < 0.05 for the mean compared to the zebrafish Refseq set. (b) Cumulative frequency histograms for the free energy of the minimum energy secondary structure (dG) for Refseq (black) and Himix (blue) gene sets. Dotted line indicates that for 90% of highly expressed genes, the dG was greater than –13.1 kcal/mol. (c) Distribution of 3′UTRs lengths (from the stop codon to the beginning of the polyadenylated sequence) in the Refseq (gray) and Himix (blue) sets. Bins sizes are 100 bp with maximum values per bin indicated on the x-axis. Inset, mean and standard error for each group. Mann–Whitney U test * P < 0.001. (d) Distribution of number of introns per gene for the Refseq (gray) and Himix (blue) sets.
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Figure 3: Structural mRNA and gene features in highly expressed genes. (a) Box plots of the maximal free energy of folding (dG) for nucleotides from −4 to +37 for genes in each data set and for genes in the mouse and human Refseq databases. *P < 0.05 for the mean compared to the zebrafish Refseq set. (b) Cumulative frequency histograms for the free energy of the minimum energy secondary structure (dG) for Refseq (black) and Himix (blue) gene sets. Dotted line indicates that for 90% of highly expressed genes, the dG was greater than –13.1 kcal/mol. (c) Distribution of 3′UTRs lengths (from the stop codon to the beginning of the polyadenylated sequence) in the Refseq (gray) and Himix (blue) sets. Bins sizes are 100 bp with maximum values per bin indicated on the x-axis. Inset, mean and standard error for each group. Mann–Whitney U test * P < 0.001. (d) Distribution of number of introns per gene for the Refseq (gray) and Himix (blue) sets.
Mentions: Translation initiation is impaired when strong secondary structure for nucleotides −4 to +37 (relative to the AUG) reduces accessibility for the ribosome (11,61). In other parts of the transcript, local mRNA structure is not generally a major determinant of the rate of translation elongation since the ribosome processively destabilizes the mRNA (62,63). The mean free energy of the minimum energy structure (dG) at 28°C for zebrafish transcripts was 2 kcal/mol greater than for mouse or human transcripts, suggesting that nucleotide sequence has adapted to reduce secondary structure at the typical environmental temperatures that zebrafish inhabit (16.5–33°C (64); Figure 3a). Highly expressed genes showed a small but significant tendency for a more open mRNA structure than other genes (Figure 3a and b).

Bottom Line: Zebrafish embryos are rapidly injected with calibrated doses of mRNA, enabling the effects of multiple sequence changes to be compared in vivo.These results suggested principles for increasing protein yield in zebrafish through biomolecular engineering.Rational gene design thus significantly boosts expression in zebrafish, and a similar approach will likely elevate expression in other animal models.

View Article: PubMed Central - PubMed

Affiliation: Program in Genomics of Differentiation, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, MD 20892, USA.

Show MeSH