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Expression of human nPTB is limited by extreme suboptimal codon content.

Robinson F, Jackson RJ, Smith CW - PLoS ONE (2008)

Bottom Line: In comparing the splicing repressor activities of transfected human PTB and its two tissue-restricted paralogs-nPTB and ROD1-we found that the three proteins were expressed at widely varying levels. nPTB was expressed at 1-3% the level of PTB despite similar levels of mRNA expression and 74% amino acid identity.We were then able to demonstrate that all three proteins act as splicing repressors.Our results provide a striking illustration of the importance of mRNA codon content in determining levels of protein expression, even within cells of the natural host species.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom.

ABSTRACT

Background: The frequency of synonymous codon usage varies widely between organisms. Suboptimal codon content limits expression of viral, experimental or therapeutic heterologous proteins due to limiting cognate tRNAs. Codon content is therefore often adjusted to match codon bias of the host organism. Codon content also varies between genes within individual mammalian species. However, little attention has been paid to the consequences of codon content upon translation of host proteins.

Methodology/principal findings: In comparing the splicing repressor activities of transfected human PTB and its two tissue-restricted paralogs-nPTB and ROD1-we found that the three proteins were expressed at widely varying levels. nPTB was expressed at 1-3% the level of PTB despite similar levels of mRNA expression and 74% amino acid identity. The low nPTB expression was due to the high proportion of codons with A or U at the third codon position, which are suboptimal in human mRNAs. Optimization of the nPTB codon content, akin to the "humanization" of foreign ORFs, allowed efficient translation in vivo and in vitro to levels comparable with PTB. We were then able to demonstrate that all three proteins act as splicing repressors.

Conclusions/significance: Our results provide a striking illustration of the importance of mRNA codon content in determining levels of protein expression, even within cells of the natural host species.

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Related in: MedlinePlus

Differential overexpression of PTB, nPTB and ROD1.A) Expression constructs featured a CMV promoter, common 5′ and 3′ UTRs and an N-terminal Xpress epitope tag. Open reading frames (ORF) for PTB and its paralogs were the only variable between constructs. nPTB* is the codon-optimized nPTB expression construct described in the text. B) Western blots of 293 cells transfected with the indicated constructs, probed with anti-Xpress (XP, upper panel) or anti-ERK antibodies. Positions of size markers (kDa) are indicated to the right. C) Western blot comparing the anti-Xpress signal obtained from a dilution series of protein from PTB1 transfected 293 cells (1.0, 0.5, 0.25, 0.062, 0.031 and 0.01 equivalents) with 1.5 equivalents from an nPTB transfection (left hand lane). D) Nuclease protection assay of cytoplasmic RNA harvested from 293 cells transfected with the constructs indicated above. 4Z is a mock transfection (pGEM4Z). “T” is the test probe, corresponding to the Xpress probe region of the expression construct, while “C” is a control probe detecting the cotransfected GFP construct. Positions of size markers (nt) are indicated to the left. E) Western blots of L cells transfected with the indicated constructs, probed with anti-Xpress (XP, upper panel) or anti-ERK antibodies. 4Z is a mock transfection (pGEM4Z). F) Nuclease protection of cytoplasmic RNA from L cells. Details as in “D”. G) Western blots of HeLa cells transfected with the indicated constructs, probed with anti-Xpress (XP, upper panel) or anti-ERK antibodies. H) Western blot comparing the anti-Xpress signal obtained from a dilution series of protein from PTB1 transfected HeLa cells (1.0, 0.5, 0.25, 0.062, 0.031 and 0.01 equivalents) with 1.5 equivalents from an nPTB transfection (left hand lane). I) Nuclease protection of cytoplasmic RNA from HeLa cells. Details as in “D”.
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pone-0001801-g001: Differential overexpression of PTB, nPTB and ROD1.A) Expression constructs featured a CMV promoter, common 5′ and 3′ UTRs and an N-terminal Xpress epitope tag. Open reading frames (ORF) for PTB and its paralogs were the only variable between constructs. nPTB* is the codon-optimized nPTB expression construct described in the text. B) Western blots of 293 cells transfected with the indicated constructs, probed with anti-Xpress (XP, upper panel) or anti-ERK antibodies. Positions of size markers (kDa) are indicated to the right. C) Western blot comparing the anti-Xpress signal obtained from a dilution series of protein from PTB1 transfected 293 cells (1.0, 0.5, 0.25, 0.062, 0.031 and 0.01 equivalents) with 1.5 equivalents from an nPTB transfection (left hand lane). D) Nuclease protection assay of cytoplasmic RNA harvested from 293 cells transfected with the constructs indicated above. 4Z is a mock transfection (pGEM4Z). “T” is the test probe, corresponding to the Xpress probe region of the expression construct, while “C” is a control probe detecting the cotransfected GFP construct. Positions of size markers (nt) are indicated to the left. E) Western blots of L cells transfected with the indicated constructs, probed with anti-Xpress (XP, upper panel) or anti-ERK antibodies. 4Z is a mock transfection (pGEM4Z). F) Nuclease protection of cytoplasmic RNA from L cells. Details as in “D”. G) Western blots of HeLa cells transfected with the indicated constructs, probed with anti-Xpress (XP, upper panel) or anti-ERK antibodies. H) Western blot comparing the anti-Xpress signal obtained from a dilution series of protein from PTB1 transfected HeLa cells (1.0, 0.5, 0.25, 0.062, 0.031 and 0.01 equivalents) with 1.5 equivalents from an nPTB transfection (left hand lane). I) Nuclease protection of cytoplasmic RNA from HeLa cells. Details as in “D”.

Mentions: In order to achieve equivalent levels of expression, the open reading frames (ORFs) for human PTB1, PTB4, nPTB and ROD1 were cloned into an expression vector that provided a common CMV promoter, 5′ and 3′ untranslated sequences and an N-terminal Xpress epitope tag (Fig 1A). The constructs were transiently co-transfected into a range of mammalian cell lines along with a GFP expression vector as a transfection control. Unexpectedly, western blotting for the Xpress epitope tag showed much lower expression of nPTB and ROD1 than of PTB1 and PTB4 in all cell lines tested (Fig. 1B,E,G). Reprobing the blots with anti-ERK antibody demonstrated the equivalence of protein loading between samples. Comparison with serial dilutions of total protein lysates from PTB1 transfections indicated that nPTB protein expression levels were 1–3% those of PTB1 in 293 and HeLa cells (Fig 1C,H). The difference in protein expression was evident at various time-points after transfection, arguing against the possibility that the paralogs might be expressed equally but fail to accumulate due to lower stability (data not shown). The possibility that overexpression of nPTB and ROD1 might be toxic to cells was ruled out by cotransfecting PTB, nPTB and ROD1 with a β-galactosidase reporter; no differences in numbers of stained cells were observed (data not shown).


Expression of human nPTB is limited by extreme suboptimal codon content.

Robinson F, Jackson RJ, Smith CW - PLoS ONE (2008)

Differential overexpression of PTB, nPTB and ROD1.A) Expression constructs featured a CMV promoter, common 5′ and 3′ UTRs and an N-terminal Xpress epitope tag. Open reading frames (ORF) for PTB and its paralogs were the only variable between constructs. nPTB* is the codon-optimized nPTB expression construct described in the text. B) Western blots of 293 cells transfected with the indicated constructs, probed with anti-Xpress (XP, upper panel) or anti-ERK antibodies. Positions of size markers (kDa) are indicated to the right. C) Western blot comparing the anti-Xpress signal obtained from a dilution series of protein from PTB1 transfected 293 cells (1.0, 0.5, 0.25, 0.062, 0.031 and 0.01 equivalents) with 1.5 equivalents from an nPTB transfection (left hand lane). D) Nuclease protection assay of cytoplasmic RNA harvested from 293 cells transfected with the constructs indicated above. 4Z is a mock transfection (pGEM4Z). “T” is the test probe, corresponding to the Xpress probe region of the expression construct, while “C” is a control probe detecting the cotransfected GFP construct. Positions of size markers (nt) are indicated to the left. E) Western blots of L cells transfected with the indicated constructs, probed with anti-Xpress (XP, upper panel) or anti-ERK antibodies. 4Z is a mock transfection (pGEM4Z). F) Nuclease protection of cytoplasmic RNA from L cells. Details as in “D”. G) Western blots of HeLa cells transfected with the indicated constructs, probed with anti-Xpress (XP, upper panel) or anti-ERK antibodies. H) Western blot comparing the anti-Xpress signal obtained from a dilution series of protein from PTB1 transfected HeLa cells (1.0, 0.5, 0.25, 0.062, 0.031 and 0.01 equivalents) with 1.5 equivalents from an nPTB transfection (left hand lane). I) Nuclease protection of cytoplasmic RNA from HeLa cells. Details as in “D”.
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Related In: Results  -  Collection

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pone-0001801-g001: Differential overexpression of PTB, nPTB and ROD1.A) Expression constructs featured a CMV promoter, common 5′ and 3′ UTRs and an N-terminal Xpress epitope tag. Open reading frames (ORF) for PTB and its paralogs were the only variable between constructs. nPTB* is the codon-optimized nPTB expression construct described in the text. B) Western blots of 293 cells transfected with the indicated constructs, probed with anti-Xpress (XP, upper panel) or anti-ERK antibodies. Positions of size markers (kDa) are indicated to the right. C) Western blot comparing the anti-Xpress signal obtained from a dilution series of protein from PTB1 transfected 293 cells (1.0, 0.5, 0.25, 0.062, 0.031 and 0.01 equivalents) with 1.5 equivalents from an nPTB transfection (left hand lane). D) Nuclease protection assay of cytoplasmic RNA harvested from 293 cells transfected with the constructs indicated above. 4Z is a mock transfection (pGEM4Z). “T” is the test probe, corresponding to the Xpress probe region of the expression construct, while “C” is a control probe detecting the cotransfected GFP construct. Positions of size markers (nt) are indicated to the left. E) Western blots of L cells transfected with the indicated constructs, probed with anti-Xpress (XP, upper panel) or anti-ERK antibodies. 4Z is a mock transfection (pGEM4Z). F) Nuclease protection of cytoplasmic RNA from L cells. Details as in “D”. G) Western blots of HeLa cells transfected with the indicated constructs, probed with anti-Xpress (XP, upper panel) or anti-ERK antibodies. H) Western blot comparing the anti-Xpress signal obtained from a dilution series of protein from PTB1 transfected HeLa cells (1.0, 0.5, 0.25, 0.062, 0.031 and 0.01 equivalents) with 1.5 equivalents from an nPTB transfection (left hand lane). I) Nuclease protection of cytoplasmic RNA from HeLa cells. Details as in “D”.
Mentions: In order to achieve equivalent levels of expression, the open reading frames (ORFs) for human PTB1, PTB4, nPTB and ROD1 were cloned into an expression vector that provided a common CMV promoter, 5′ and 3′ untranslated sequences and an N-terminal Xpress epitope tag (Fig 1A). The constructs were transiently co-transfected into a range of mammalian cell lines along with a GFP expression vector as a transfection control. Unexpectedly, western blotting for the Xpress epitope tag showed much lower expression of nPTB and ROD1 than of PTB1 and PTB4 in all cell lines tested (Fig. 1B,E,G). Reprobing the blots with anti-ERK antibody demonstrated the equivalence of protein loading between samples. Comparison with serial dilutions of total protein lysates from PTB1 transfections indicated that nPTB protein expression levels were 1–3% those of PTB1 in 293 and HeLa cells (Fig 1C,H). The difference in protein expression was evident at various time-points after transfection, arguing against the possibility that the paralogs might be expressed equally but fail to accumulate due to lower stability (data not shown). The possibility that overexpression of nPTB and ROD1 might be toxic to cells was ruled out by cotransfecting PTB, nPTB and ROD1 with a β-galactosidase reporter; no differences in numbers of stained cells were observed (data not shown).

Bottom Line: In comparing the splicing repressor activities of transfected human PTB and its two tissue-restricted paralogs-nPTB and ROD1-we found that the three proteins were expressed at widely varying levels. nPTB was expressed at 1-3% the level of PTB despite similar levels of mRNA expression and 74% amino acid identity.We were then able to demonstrate that all three proteins act as splicing repressors.Our results provide a striking illustration of the importance of mRNA codon content in determining levels of protein expression, even within cells of the natural host species.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom.

ABSTRACT

Background: The frequency of synonymous codon usage varies widely between organisms. Suboptimal codon content limits expression of viral, experimental or therapeutic heterologous proteins due to limiting cognate tRNAs. Codon content is therefore often adjusted to match codon bias of the host organism. Codon content also varies between genes within individual mammalian species. However, little attention has been paid to the consequences of codon content upon translation of host proteins.

Methodology/principal findings: In comparing the splicing repressor activities of transfected human PTB and its two tissue-restricted paralogs-nPTB and ROD1-we found that the three proteins were expressed at widely varying levels. nPTB was expressed at 1-3% the level of PTB despite similar levels of mRNA expression and 74% amino acid identity. The low nPTB expression was due to the high proportion of codons with A or U at the third codon position, which are suboptimal in human mRNAs. Optimization of the nPTB codon content, akin to the "humanization" of foreign ORFs, allowed efficient translation in vivo and in vitro to levels comparable with PTB. We were then able to demonstrate that all three proteins act as splicing repressors.

Conclusions/significance: Our results provide a striking illustration of the importance of mRNA codon content in determining levels of protein expression, even within cells of the natural host species.

Show MeSH
Related in: MedlinePlus