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Translational control of recombinant human acetylcholinesterase accumulation in plants.

Geyer BC, Fletcher SP, Griffin TA, Lopker MJ, Soreq H, Mor TS - BMC Biotechnol. (2007)

Bottom Line: We have challenged this conclusion by expressing the human acetylcholinesterase coding sequence in transgenic plants in its native GC-rich sequence and compared to a matched sequence with (dicotyledonous) plant-optimized codon usage and a lower GC content.We demonstrate a 5 to 10 fold increase in accumulation levels of the "synaptic" splice variant of human acetylcholinesterase in Nicotiana benthamiana plants expressing the optimized gene as compared to the native human sequence.Importantly, we find that the increase is not a result of increased levels of acetylcholinesterase mRNA, but rather its facilitated translation, possibly due to the reduced energy required to unfold the sequence-optimized mRNA.

View Article: PubMed Central - HTML - PubMed

Affiliation: The School of Life Sciences and Biodesign Institute, Arizona State University, Tempe, AZ 85287-4501, USA. bgeyer@mainex1.asu.edu <bgeyer@mainex1.asu.edu>

ABSTRACT

Background: Codon usage differences are known to regulate the levels of gene expression in a species-specific manner, with the primary factors often cited to be mRNA processing and accumulation. We have challenged this conclusion by expressing the human acetylcholinesterase coding sequence in transgenic plants in its native GC-rich sequence and compared to a matched sequence with (dicotyledonous) plant-optimized codon usage and a lower GC content.

Results: We demonstrate a 5 to 10 fold increase in accumulation levels of the "synaptic" splice variant of human acetylcholinesterase in Nicotiana benthamiana plants expressing the optimized gene as compared to the native human sequence. Both transient expression assays and stable transformants demonstrated conspicuously increased accumulation levels. Importantly, we find that the increase is not a result of increased levels of acetylcholinesterase mRNA, but rather its facilitated translation, possibly due to the reduced energy required to unfold the sequence-optimized mRNA.

Conclusion: Our findings demonstrate that codon usage differences may regulate gene expression at different levels and anticipate translational control of acetylcholinesterase gene expression in its native mammalian host as well.

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Sequence optimization of the hACHE-S gene. Shown are the native (cDNA) human gene sequence (upper) and the optimized gene (lower). Nucleotide changes are shown, whereas unmodified nucleotides are represented by dashes. Above the two compared DNA sequences is the predicted amino-acid sequence of the AChE-S protein. Underline: potential methylation sites. Yellow box: infrequent plant codons. Red box: potential 5'-intron splice sites. Blue box: potential downstream 3'-intron splice sites. Purple box: a potential polyadenylation positional element. Please note that the only amino-acid deviations from the human cDNA are an R2A change in the human sequence and insertion of a G residue on this position in the plant-optimized sequence. These changes were introduced to facilitate cloning into the plant expression vectors used in this study.
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Figure 1: Sequence optimization of the hACHE-S gene. Shown are the native (cDNA) human gene sequence (upper) and the optimized gene (lower). Nucleotide changes are shown, whereas unmodified nucleotides are represented by dashes. Above the two compared DNA sequences is the predicted amino-acid sequence of the AChE-S protein. Underline: potential methylation sites. Yellow box: infrequent plant codons. Red box: potential 5'-intron splice sites. Blue box: potential downstream 3'-intron splice sites. Purple box: a potential polyadenylation positional element. Please note that the only amino-acid deviations from the human cDNA are an R2A change in the human sequence and insertion of a G residue on this position in the plant-optimized sequence. These changes were introduced to facilitate cloning into the plant expression vectors used in this study.

Mentions: More than a third of the codons of the human ACHE gene (hACHE-S) are infrequently utilized in dicotyledonous plants (i.e. as defined in the Methods section, they have relative synonymous codon usage, RSCU, values of less than 0.8, Fig. 1, Fig. 2, Table 1). Furthermore, a non-canonical near-upstream element of a plant polyadenylation signal [24,25] (Fig. 2, Table 1) and two plant 5'-intron splice signals with downstream 3'-splice signals were identified in hACHES [26,27] (Fig. 2, Table 1). In addition, potential methylation signals associated with transcriptional silencing (CG and CNG) [28] are abundant in the gene, reflecting its high GC content (65%, Fig. 1, Fig. 2, Table 1). Interestingly, the GC content in mid-exonic regions is even higher, with peaks exceeding 75% (Fig. 2). These regions are separated by deep troughs with much lower GC content (<50%). In designing a plant-expression optimized version of the gene, we took measures to correct for these potential problems. Thus, the rare codons present in hACHE-S were changed in the synthetic plant-expression optimized ACHE gene (oACHES) to more frequently used alternatives so that the codon adaptiveness index (CAI, for definition see the Methods section) would match that of the most abundant nuclear-encoded plant protein – the small subunit of ribulose bisphosphate carboxylase (RuBisCO, Fig. 2 and Table 1). Furthermore, the RNA processing signals were abolished, most of the potential methylation signals were eliminated, and the overall GC content was reduced to 55%, much closer to the typical plant gene (Fig. 2, Table 1). In particular, most of the GC content peaks were considerably dampened. Please note that our optimization strategy did not eliminate all infrequently used codons, giving preference to the elimination of all other deleterious sequences. However, the content of such (evenly distributed) codons was reduced to about 20% (similar to the case of the RuBisCO small subunit, Fig. 2, Table 1). When cloning hACHE-S and oACHE-S into plant expression vectors, two minor changes in the amino acid sequence of the encoded proteins were required due to the need to introduce an NcoI site to facilitate the cloning (Fig. 1). These deviations from the human sequence (R2A in hACHE-S and insertion of G between M1 and R2 in oACHE-S) are both in the cleavable signal peptide directing the protein to the secretory pathway.


Translational control of recombinant human acetylcholinesterase accumulation in plants.

Geyer BC, Fletcher SP, Griffin TA, Lopker MJ, Soreq H, Mor TS - BMC Biotechnol. (2007)

Sequence optimization of the hACHE-S gene. Shown are the native (cDNA) human gene sequence (upper) and the optimized gene (lower). Nucleotide changes are shown, whereas unmodified nucleotides are represented by dashes. Above the two compared DNA sequences is the predicted amino-acid sequence of the AChE-S protein. Underline: potential methylation sites. Yellow box: infrequent plant codons. Red box: potential 5'-intron splice sites. Blue box: potential downstream 3'-intron splice sites. Purple box: a potential polyadenylation positional element. Please note that the only amino-acid deviations from the human cDNA are an R2A change in the human sequence and insertion of a G residue on this position in the plant-optimized sequence. These changes were introduced to facilitate cloning into the plant expression vectors used in this study.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1913049&req=5

Figure 1: Sequence optimization of the hACHE-S gene. Shown are the native (cDNA) human gene sequence (upper) and the optimized gene (lower). Nucleotide changes are shown, whereas unmodified nucleotides are represented by dashes. Above the two compared DNA sequences is the predicted amino-acid sequence of the AChE-S protein. Underline: potential methylation sites. Yellow box: infrequent plant codons. Red box: potential 5'-intron splice sites. Blue box: potential downstream 3'-intron splice sites. Purple box: a potential polyadenylation positional element. Please note that the only amino-acid deviations from the human cDNA are an R2A change in the human sequence and insertion of a G residue on this position in the plant-optimized sequence. These changes were introduced to facilitate cloning into the plant expression vectors used in this study.
Mentions: More than a third of the codons of the human ACHE gene (hACHE-S) are infrequently utilized in dicotyledonous plants (i.e. as defined in the Methods section, they have relative synonymous codon usage, RSCU, values of less than 0.8, Fig. 1, Fig. 2, Table 1). Furthermore, a non-canonical near-upstream element of a plant polyadenylation signal [24,25] (Fig. 2, Table 1) and two plant 5'-intron splice signals with downstream 3'-splice signals were identified in hACHES [26,27] (Fig. 2, Table 1). In addition, potential methylation signals associated with transcriptional silencing (CG and CNG) [28] are abundant in the gene, reflecting its high GC content (65%, Fig. 1, Fig. 2, Table 1). Interestingly, the GC content in mid-exonic regions is even higher, with peaks exceeding 75% (Fig. 2). These regions are separated by deep troughs with much lower GC content (<50%). In designing a plant-expression optimized version of the gene, we took measures to correct for these potential problems. Thus, the rare codons present in hACHE-S were changed in the synthetic plant-expression optimized ACHE gene (oACHES) to more frequently used alternatives so that the codon adaptiveness index (CAI, for definition see the Methods section) would match that of the most abundant nuclear-encoded plant protein – the small subunit of ribulose bisphosphate carboxylase (RuBisCO, Fig. 2 and Table 1). Furthermore, the RNA processing signals were abolished, most of the potential methylation signals were eliminated, and the overall GC content was reduced to 55%, much closer to the typical plant gene (Fig. 2, Table 1). In particular, most of the GC content peaks were considerably dampened. Please note that our optimization strategy did not eliminate all infrequently used codons, giving preference to the elimination of all other deleterious sequences. However, the content of such (evenly distributed) codons was reduced to about 20% (similar to the case of the RuBisCO small subunit, Fig. 2, Table 1). When cloning hACHE-S and oACHE-S into plant expression vectors, two minor changes in the amino acid sequence of the encoded proteins were required due to the need to introduce an NcoI site to facilitate the cloning (Fig. 1). These deviations from the human sequence (R2A in hACHE-S and insertion of G between M1 and R2 in oACHE-S) are both in the cleavable signal peptide directing the protein to the secretory pathway.

Bottom Line: We have challenged this conclusion by expressing the human acetylcholinesterase coding sequence in transgenic plants in its native GC-rich sequence and compared to a matched sequence with (dicotyledonous) plant-optimized codon usage and a lower GC content.We demonstrate a 5 to 10 fold increase in accumulation levels of the "synaptic" splice variant of human acetylcholinesterase in Nicotiana benthamiana plants expressing the optimized gene as compared to the native human sequence.Importantly, we find that the increase is not a result of increased levels of acetylcholinesterase mRNA, but rather its facilitated translation, possibly due to the reduced energy required to unfold the sequence-optimized mRNA.

View Article: PubMed Central - HTML - PubMed

Affiliation: The School of Life Sciences and Biodesign Institute, Arizona State University, Tempe, AZ 85287-4501, USA. bgeyer@mainex1.asu.edu <bgeyer@mainex1.asu.edu>

ABSTRACT

Background: Codon usage differences are known to regulate the levels of gene expression in a species-specific manner, with the primary factors often cited to be mRNA processing and accumulation. We have challenged this conclusion by expressing the human acetylcholinesterase coding sequence in transgenic plants in its native GC-rich sequence and compared to a matched sequence with (dicotyledonous) plant-optimized codon usage and a lower GC content.

Results: We demonstrate a 5 to 10 fold increase in accumulation levels of the "synaptic" splice variant of human acetylcholinesterase in Nicotiana benthamiana plants expressing the optimized gene as compared to the native human sequence. Both transient expression assays and stable transformants demonstrated conspicuously increased accumulation levels. Importantly, we find that the increase is not a result of increased levels of acetylcholinesterase mRNA, but rather its facilitated translation, possibly due to the reduced energy required to unfold the sequence-optimized mRNA.

Conclusion: Our findings demonstrate that codon usage differences may regulate gene expression at different levels and anticipate translational control of acetylcholinesterase gene expression in its native mammalian host as well.

Show MeSH
Related in: MedlinePlus