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Optimization of mRNA design for protein expression in the crustacean Daphnia magna

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Abstract

The water flea Daphnia is a new model organism for ecological, evolutionary, and toxicological genomics. Detailed functional analysis of genes newly discovered through genomic approaches often requires overexpression of the identified protein. In the present study, we report the microinjection of in vitro-synthesized RNAs into the eggs as a method for overexpressing ubiquitous proteins in Daphnia magna. We injected a 1.3-kb mRNA that coded for the red fluorescent protein (DsRed2) flanked by UTRs from the ubiquitously expressed elongation factor 1α-1 (EF1α-1) into D. magna embryos. DsRed2 fluorescence in the embryos was measured 24 h after microinjection. Unexpectedly, the reporter RNA containing the 522-bp full-length EF1α-1 3′ UTR failed to induce fluorescence. To assess reporter expression, the length of the 3′ UTR that potentially contained negative regulatory elements of protein expression, including AU-rich regions and Musashi binding elements, was serially reduced from the 3′ end. Assessing all injected RNA alternatives, mRNA containing the first 60 bp of the 3′ UTR gave rise to the highest fluorescence, 14 times the Daphnia auto-fluorescence. In contrast, mRNA lacking the entire 3′ UTR hardly induced any change in fluorescence intensity. This is the first evaluation of UTRs of mRNAs delivered into Daphnia embryos by microinjection for overexpressing proteins. The mRNA with truncated 3′ UTRs of Daphnia EF1α-1 will be useful not only for gain-of-function analyses but also for labeling proteins and organelles with fluorescent proteins in Daphnia.

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Acknowledgments

We thank Dr. Minoru Tanaka of National Institute for Basic Biology, Japan, for providing the pRCS21 vector. This study was supported by a grant from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) and partially supported by the Asahi Glass Foundation. One of the authors (Y.K.) would like to acknowledge the support from the Frontier Research Base for Global Young Researchers, Osaka University, based on the Program of MEXT.

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Authors and Affiliations

  1. Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka, 565-0871, Japan

    Kerstin Törner, Takashi Nakanishi, Tomoaki Matsuura, Yasuhiko Kato & Hajime Watanabe

  2. Frontier Research Base for Global Young Researchers, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka, 565-0871, Japan

    Yasuhiko Kato

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  1. Kerstin Törner
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  2. Takashi Nakanishi
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  3. Tomoaki Matsuura
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  4. Yasuhiko Kato
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  5. Hajime Watanabe
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Correspondence to Hajime Watanabe.

Additional information

Communicated by S. Hohmann.

K. Törner and T. Nakanishi contributed equally to this work.

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438_2014_830_MOESM1_ESM.eps

Supplementary Fig. 1 Sequence of chimeric DsRed2 DNA. Nucleotide sequence of template DNA for RNA synthesis. SP6 promoter sequence is shown in dark blue, Xenopus β-globin leader sequence in brown, EF1α-1 5′ UTR in red, DsRed2 ORF in green, and EF1α-1 3′ UTR in light blue. Kozak sequence and the first 60 nt of the 3′ UTR are marked with black boxes (EPS 6480 kb)

438_2014_830_MOESM2_ESM.eps

Supplementary Fig. 2 Comparison of EF1α 3′ UTRs from different organisms. EF1α 3′ UTRs from different organisms and their percentage CG content. AUUUA motifs are indicated by red arrowheads (EPS 6033 kb)

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Törner, K., Nakanishi, T., Matsuura, T. et al. Optimization of mRNA design for protein expression in the crustacean Daphnia magna . Mol Genet Genomics 289, 707–715 (2014). https://doi.org/10.1007/s00438-014-0830-8

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