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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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Related in: MedlinePlus

Full-length hACHE-S and oACHE-S transcripts accumulate to similar levels. (A) RT-PCR amplification of ACHE transcript region potentially subjected to aberrant splicing. cDNA prepared from three lines of hACHE-S-R177W plants (1) and two lines of oACHE-S plants (2) were subjected to PCR amplification using the primers oTM089 and oTM049 and then resolved by agarose gel electrophoresis. Primers were selected to amplify the region potentially subjected to alternative splicing. Unspliced transcripts are expected to yield 1.1 kb fragment. If either of the two potential 5'- and 3'-intron splice sites were extensively utilized in plants harboring hACHE-S, shorter fragments of ~0.9 kb and ~0.5 kb are expected to be preferentially amplified. However, the full-length species clearly dominates. (B-C) Correlation between ACHE transcript accumulation and AChE enzyme activity. Transcripts levels of optimized (pTM092) and non-optimized (pTM050) measured by quantitative real time RT-PCR and were scored relative to 18S rRNA. Probes were designed for the two common regions of the two types of ACHE transcripts – (B) the 5'-UTR (TEV leader) and (C) the 3'-UTR (VSP terminator, upstream of the poly-A tail). Data obtained for multiple replicates was averaged and correlated with the respective AChE activity (normalized to the maximum level). When probed by the 5'-UTR probe, the correlation was highly significant with r2 = 0.72 (P < 0.0001) for the non-optimized sequence and r2 = 0.61 (P = 0.0005) for the optimized sequence. When probed by the 3'-UTR probe, the correlation was significant for the non-optimized sequence with r2 = 0.48 (P = 0.0122) and highly significant with r2 = 0.49 (P < 0.0001) for the optimized sequence. Inserts show scatter plots demonstrating the distribution of transcript levels in plants expressing hACHE-S and oACHE-S constructs. Each plant is depicted by a symbol and the mean ± S.D. are plotted. The groups are not statistically different by an unpaired t test (5'-UTR probe: P > 0.45; 3'-UTR probe: P > 0.22).
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Figure 4: Full-length hACHE-S and oACHE-S transcripts accumulate to similar levels. (A) RT-PCR amplification of ACHE transcript region potentially subjected to aberrant splicing. cDNA prepared from three lines of hACHE-S-R177W plants (1) and two lines of oACHE-S plants (2) were subjected to PCR amplification using the primers oTM089 and oTM049 and then resolved by agarose gel electrophoresis. Primers were selected to amplify the region potentially subjected to alternative splicing. Unspliced transcripts are expected to yield 1.1 kb fragment. If either of the two potential 5'- and 3'-intron splice sites were extensively utilized in plants harboring hACHE-S, shorter fragments of ~0.9 kb and ~0.5 kb are expected to be preferentially amplified. However, the full-length species clearly dominates. (B-C) Correlation between ACHE transcript accumulation and AChE enzyme activity. Transcripts levels of optimized (pTM092) and non-optimized (pTM050) measured by quantitative real time RT-PCR and were scored relative to 18S rRNA. Probes were designed for the two common regions of the two types of ACHE transcripts – (B) the 5'-UTR (TEV leader) and (C) the 3'-UTR (VSP terminator, upstream of the poly-A tail). Data obtained for multiple replicates was averaged and correlated with the respective AChE activity (normalized to the maximum level). When probed by the 5'-UTR probe, the correlation was highly significant with r2 = 0.72 (P < 0.0001) for the non-optimized sequence and r2 = 0.61 (P = 0.0005) for the optimized sequence. When probed by the 3'-UTR probe, the correlation was significant for the non-optimized sequence with r2 = 0.48 (P = 0.0122) and highly significant with r2 = 0.49 (P < 0.0001) for the optimized sequence. Inserts show scatter plots demonstrating the distribution of transcript levels in plants expressing hACHE-S and oACHE-S constructs. Each plant is depicted by a symbol and the mean ± S.D. are plotted. The groups are not statistically different by an unpaired t test (5'-UTR probe: P > 0.45; 3'-UTR probe: P > 0.22).

Mentions: To corroborate this conclusion, we amplified by PCR cDNA derived from either pTM050 (harboring hACHE-S with a single base change) or pTM092 (oACHE-S) lines using primers specific to the region encompassing the potential introns. In both cases the only fragments amplified corresponded to the correct, full length, sequence (Fig. 4A). Therefore, it seems unlikely that spurious splicing events are responsible for the poor performance of the hACHE-S construct. Similarly, an RNA blot analysis using probes specific for the human vs. plant-expression optimized sequences, demonstrated single, identically sized transcripts in both hACHE-S and oACHE-S of the predicted length (data not shown), abating the probability that premature polyadenylation was limiting hACHE-S expression as compared to oACHE-S.


Translational control of recombinant human acetylcholinesterase accumulation in plants.

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

Full-length hACHE-S and oACHE-S transcripts accumulate to similar levels. (A) RT-PCR amplification of ACHE transcript region potentially subjected to aberrant splicing. cDNA prepared from three lines of hACHE-S-R177W plants (1) and two lines of oACHE-S plants (2) were subjected to PCR amplification using the primers oTM089 and oTM049 and then resolved by agarose gel electrophoresis. Primers were selected to amplify the region potentially subjected to alternative splicing. Unspliced transcripts are expected to yield 1.1 kb fragment. If either of the two potential 5'- and 3'-intron splice sites were extensively utilized in plants harboring hACHE-S, shorter fragments of ~0.9 kb and ~0.5 kb are expected to be preferentially amplified. However, the full-length species clearly dominates. (B-C) Correlation between ACHE transcript accumulation and AChE enzyme activity. Transcripts levels of optimized (pTM092) and non-optimized (pTM050) measured by quantitative real time RT-PCR and were scored relative to 18S rRNA. Probes were designed for the two common regions of the two types of ACHE transcripts – (B) the 5'-UTR (TEV leader) and (C) the 3'-UTR (VSP terminator, upstream of the poly-A tail). Data obtained for multiple replicates was averaged and correlated with the respective AChE activity (normalized to the maximum level). When probed by the 5'-UTR probe, the correlation was highly significant with r2 = 0.72 (P < 0.0001) for the non-optimized sequence and r2 = 0.61 (P = 0.0005) for the optimized sequence. When probed by the 3'-UTR probe, the correlation was significant for the non-optimized sequence with r2 = 0.48 (P = 0.0122) and highly significant with r2 = 0.49 (P < 0.0001) for the optimized sequence. Inserts show scatter plots demonstrating the distribution of transcript levels in plants expressing hACHE-S and oACHE-S constructs. Each plant is depicted by a symbol and the mean ± S.D. are plotted. The groups are not statistically different by an unpaired t test (5'-UTR probe: P > 0.45; 3'-UTR probe: P > 0.22).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Full-length hACHE-S and oACHE-S transcripts accumulate to similar levels. (A) RT-PCR amplification of ACHE transcript region potentially subjected to aberrant splicing. cDNA prepared from three lines of hACHE-S-R177W plants (1) and two lines of oACHE-S plants (2) were subjected to PCR amplification using the primers oTM089 and oTM049 and then resolved by agarose gel electrophoresis. Primers were selected to amplify the region potentially subjected to alternative splicing. Unspliced transcripts are expected to yield 1.1 kb fragment. If either of the two potential 5'- and 3'-intron splice sites were extensively utilized in plants harboring hACHE-S, shorter fragments of ~0.9 kb and ~0.5 kb are expected to be preferentially amplified. However, the full-length species clearly dominates. (B-C) Correlation between ACHE transcript accumulation and AChE enzyme activity. Transcripts levels of optimized (pTM092) and non-optimized (pTM050) measured by quantitative real time RT-PCR and were scored relative to 18S rRNA. Probes were designed for the two common regions of the two types of ACHE transcripts – (B) the 5'-UTR (TEV leader) and (C) the 3'-UTR (VSP terminator, upstream of the poly-A tail). Data obtained for multiple replicates was averaged and correlated with the respective AChE activity (normalized to the maximum level). When probed by the 5'-UTR probe, the correlation was highly significant with r2 = 0.72 (P < 0.0001) for the non-optimized sequence and r2 = 0.61 (P = 0.0005) for the optimized sequence. When probed by the 3'-UTR probe, the correlation was significant for the non-optimized sequence with r2 = 0.48 (P = 0.0122) and highly significant with r2 = 0.49 (P < 0.0001) for the optimized sequence. Inserts show scatter plots demonstrating the distribution of transcript levels in plants expressing hACHE-S and oACHE-S constructs. Each plant is depicted by a symbol and the mean ± S.D. are plotted. The groups are not statistically different by an unpaired t test (5'-UTR probe: P > 0.45; 3'-UTR probe: P > 0.22).
Mentions: To corroborate this conclusion, we amplified by PCR cDNA derived from either pTM050 (harboring hACHE-S with a single base change) or pTM092 (oACHE-S) lines using primers specific to the region encompassing the potential introns. In both cases the only fragments amplified corresponded to the correct, full length, sequence (Fig. 4A). Therefore, it seems unlikely that spurious splicing events are responsible for the poor performance of the hACHE-S construct. Similarly, an RNA blot analysis using probes specific for the human vs. plant-expression optimized sequences, demonstrated single, identically sized transcripts in both hACHE-S and oACHE-S of the predicted length (data not shown), abating the probability that premature polyadenylation was limiting hACHE-S expression as compared to oACHE-S.

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