A positive readout single transcript reporter for site-specific mRNA cleavage
- Creators
- Kandul, Nikolay
- Guo, Ming
- Hay, Bruce A.
Abstract
Cleavage of mRNA molecules causes their rapid degradation, thereby playing an important role in regulation of gene expression and host genome defense from viruses and transposons in bacterial and eukaryotic cells. Current negative-readout, and repressor-based positive-readout reporters of mRNA degradation have limitations. Here we report the development of a single transcript that acts as a positive reporter of mRNA cleavage. We show that placement of bacterial CopT and CopA hairpins into the 5′ UTR and 3′ UTR of an mRNA results in inhibition of translation of the intervening coding sequence in Drosophila. An internal poly(A) tract inserted downstream of the coding sequence stabilizes transcripts cut within the 3′ UTR. When these components are combined in a transcript in which targets sites for RNA cleavage are placed between the poly(A) tract and CopA, cleavage results in translational activation, providing a single transcript-based method of sensing mRNA cleavage with a positive readout.
Additional Information
© 2017 Kandul et al. Distributed under Creative Commons CC-BY 4.0 Published July 20, 2017. The authors declare there are no competing interests. Author Contributions: Nikolay Kandul conceived and designed the experiments, performed the experiments, analyzed the data, contributed reagents/materials/analysis tools, wrote the paper, prepared figures and/or tables, reviewed drafts of the paper. Ming Guo conceived and designed the experiments, wrote the paper, reviewed drafts of the paper. Bruce A. Hay conceived and designed the experiments, analyzed the data, contributed reagents/materials/analysis tools, wrote the paper, reviewed drafts of the paper. DNA Deposition: The following information was supplied regarding the deposition of DNA sequences: Sequences are available in GenBank (KY412813, KY412814, and KY412815) and in the Supplemental Information. Data Availability: The following information was supplied regarding data availability: The raw data are located in the Supplementary Materials. This work was supported by a Caltech Alcott fellowship to NPK, by grants from the United States Department of Agriculture, National Institute for Food and Agriculture (USDA-NIFA) and the Citrus Research and Development Foundation to BAH, and by grants to MG from NIH. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Attached Files
Published - peerj-3602.pdf
Supplemental Material - GenBank_Sequences.zip
Supplemental Material - SupplementaryTables.pdf
Files
Additional details
- PMCID
- PMC5522606
- Eprint ID
- 79587
- Resolver ID
- CaltechAUTHORS:20170731-095152425
- Caltech
- US Department of Agriculture
- National Institute for Food and Agriculture
- Citrus Research and Development Foundation
- NIH
- Created
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2017-08-01Created from EPrint's datestamp field
- Updated
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2023-06-02Created from EPrint's last_modified field