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A positive readout single transcript reporter for site-specific mRNA cleavage

Kandul, Nikolay and Guo, Ming and Hay, Bruce A. (2017) A positive readout single transcript reporter for site-specific mRNA cleavage. PeerJ, 5 . Art. No. e3602. ISSN 2167-8359. PMCID PMC5522606. https://resolver.caltech.edu/CaltechAUTHORS:20170731-095152425

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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.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.7717/peerj.3602DOIArticle
https://peerj.com/articles/3602/PublisherArticle
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5522606PubMed CentralArticle
https://doi.org/10.7717/peerj.3602/supp-1DOISupplementary Tables
https://doi.org/10.7717/peerj.3602/supp-2DOIPlasmid sequences
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.
Funders:
Funding AgencyGrant Number
CaltechUNSPECIFIED
US Department of AgricultureUNSPECIFIED
National Institute for Food and AgricultureUNSPECIFIED
Citrus Research and Development FoundationUNSPECIFIED
NIHUNSPECIFIED
Subject Keywords:Poly(A), Translation, Positive-readout, RNA hairpins, CopT-CopA, Auto-repression, Reporter
PubMed Central ID:PMC5522606
Record Number:CaltechAUTHORS:20170731-095152425
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20170731-095152425
Official Citation:Kandul N, Guo M, Hay BA. (2017) A positive readout single transcript reporter for site-specific mRNA cleavage. PeerJ 5:e3602 https://doi.org/10.7717/peerj.3602
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:79587
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:01 Aug 2017 19:51
Last Modified:03 Oct 2019 18:21

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