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Biosynthetic labeling of RNA with uracil phosphoribosyltransferase allows cell-specific microarray analysis of mRNA synthesis and decay

Nature Biotechnology volume 23, pages 232–237 (2005)Cite this article

Abstract

Standard microarrays measure mRNA abundance, not mRNA synthesis, and therefore cannot identify the mechanisms that regulate gene expression. We have developed a method to overcome this limitation by using the salvage enzyme uracil phosphoribosyltransferase (UPRT) from the protozoan Toxoplasma gondii. T. gondii UPRT has been well characterized because of its application in monitoring parasite growth: mammals lack this enzyme activity and thus only the parasite incorporates 3H-uracil into its nucleic acids1,2. In this study we used RNA labeling by UPRT to determine the roles of mRNA synthesis and decay in the control of gene expression during T. gondii asexual development. We also used this approach to specifically label parasite RNA during a mouse infection and to incorporate thio-substituted uridines into the RNA of human cells engineered to express T. gondii UPRT, indicating that engineered UPRT expression will allow cell-specific analysis of gene expression in organisms other than T. gondii.

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Figure 1: sU-labeling of T. gondii RNA (a) T. gondii growth measured as the number of parasites per vacuole following growth in either 2 mM DTU (in DMSO) or an equivalent concentration of DMSO; average and s.d. from two experiments.
Figure 2: Comparison of mRNA synthesis and abundance reveals regulation of gene expression that is not detectable by traditional microarrays.
Figure 3: Relative mRNA decay measured by pulse-chase.
Figure 4: sU-labeling of RNA in T. gondii-UPRT transgenic HeLa cells and sU-labeling in vivo.

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Acknowledgements

We thank Edward Mocarski, James McCloskey, Dan Herschlag and members of the Boothroyd lab for helpful discussions. Jon Boyle provided the S23 parasite strain and technical assistance with the mouse infection experiments. M.D.C. was supported by the National Institutes of Health (NIH) (CMB GM07276) and the University of California University-wide AIDS Research Program (D02-ST-405). C.D.M. was supported by the NIH (5T32AI07328 and 1F32AI056959). E.J. was supported by the Damon Runyon Cancer Research Foundation (DRG-1630). R.G. was supported by the NIH (GM29812). J.C.B. was supported by the NIH (AI41014 and AI21423).

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Authors and Affiliations

  1. Department of Microbiology and Immunology, Stanford University School of Medicine, 299 Campus Dr., Stanford, 94305-5124, California, USA

    Michael D Cleary, Christopher D Meiering, Eric Jan & John C Boothroyd

  2. Department of Medicinal Chemistry, University of Utah, 30 S. 2000 E., Rm. 311A, Salt Lake City, 84112-5820, Utah, USA

    Rebecca Guymon

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Correspondence to John C Boothroyd.

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Supplementary information

Supplementary Fig. 1

Identification of 4-thiouridine in purified sU-labeled mRNA. (PDF 132 kb)

Supplementary Fig. 2

Normalization of pulse-chase microarrays (PDF 154 kb)

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Cleary, M., Meiering, C., Jan, E. et al. Biosynthetic labeling of RNA with uracil phosphoribosyltransferase allows cell-specific microarray analysis of mRNA synthesis and decay. Nat Biotechnol 23, 232–237 (2005). https://doi.org/10.1038/nbt1061

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