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Transcripts of a heterologous gene encoding mite allergen Der f 7 are stabilized by codon optimization in Aspergillus oryzae

  • Applied genetics and molecular biotechnology
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Abstract

We have previously demonstrated that transcripts of an AT-biased heterologous gene encoding mite allergen Der f 7 from Dermatophagoides farinae were polyadenylated prematurely within the coding region when native cDNA was expressed in Aspergillus oryzae, and that this premature polyadenylation was prevented by the codon optimization of the Der f 7 gene, resulting in increased steady-state mRNA levels. In this study, we tested the stability of transcription products derived from expression constructs of the native and codon-optimized Der f 7 gene in A. oryzae using 1,10-phenanthroline as a transcription inhibitor. Transcription products of native Der f 7 cDNA fused to the A. oryzae glucoamylase gene (glaA) were rapidly degraded; the half-life of the mRNA was approximately 13 min. However, the half-life of codon-optimized Der f 7 mRNA fused to glaA was approximately 43 min, which was highly similar to that of endogenous glaA mRNA. These results indicate that Der f 7 mRNA is significantly stabilized by codon optimization. In addition, Der f 7 mRNA was stabilized by the codon optimization of only the 3′-half region, where premature polyadenylation sites were exclusively situated; the half-life of the chimeric Der f 7 mRNA was approximately 39 min. This suggested that destabilization of native Der f 7 mRNA is mainly triggered by premature polyadenylation within the coding region. To the best of our knowledge, this is the first report to provide experimental evidence that heterologous mRNA is significantly stabilized by codon optimization in eukaryotic cells.

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Acknowledgments

We thank Natsumi Okada for the DNA sequencing. This work was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas, Applied Genomics (no. 17019001), from the Ministry of Education, Culture, Sports, Science and Technology of Japan. M. Tanaka was the recipient of a JSPS Fellowship for Japanese Young Scientists.

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Author notes

  1. Masafumi Tokuoka

    Present address: National Research Institute of Brewing, 3-7-1 Kagamiyama, Higashi-Hiroshima, Hiroshima, 739-0046, Japan

Authors and Affiliations

  1. Laboratory of Bioindustrial Genomics, Department of Bioindustrial Informatics and Genomics, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai, 981-8555, Japan

    Mizuki Tanaka, Masafumi Tokuoka, Takahiro Shintani & Katsuya Gomi

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  1. Mizuki Tanaka
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  2. Masafumi Tokuoka
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  3. Takahiro Shintani
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Correspondence to Katsuya Gomi.

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ESM 1

3′-RACE analysis of Der f 7 cDNA using total RNA prepared from mycelia treated with 1,10-phenanthroline for 20 min. a Analysis of the inserted fragments of 3′-RACE clones. First-strand cDNA was synthesized from the total RNA that was used for Northern blot analysis in Fig. 2 (time 0). Amplified PCR fragments of Der f 7 cDNA were cloned into the pCRII TA-cloning vector, as described previously (Tokuoka et al. 2008). The resulting plasmid DNAs were digested with EcoRI, followed by agarose gel electrophoresis. The arrow indicates the bands of the TA-cloning vector used for cloning of the RT-PCR products, while the arrowhead indicates the molecular marker of 500 bp. b Polyadenylation sites of native Der f 7 mRNA determined by the sequencing of 3′-RACE clones. The nucleotide sequence of the coding region is indicated in uppercase letters, whereas the terminator sequence is indicated in lowercase letters. Arrows indicate the polyadenylation sites, while the numbers beside the arrows indicate the number of clones containing a poly(A) tract at the indicated position. (PDF 214 kb)

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Tanaka, M., Tokuoka, M., Shintani, T. et al. Transcripts of a heterologous gene encoding mite allergen Der f 7 are stabilized by codon optimization in Aspergillus oryzae . Appl Microbiol Biotechnol 96, 1275–1282 (2012). https://doi.org/10.1007/s00253-012-4169-y

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