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

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.

References

  1. Pfefferkorn, E.R. & Pfefferkorn, L.C. Specific labeling of intracellular Toxoplasma gondii with uracil. J. Protozool. 24, 449–453 (1977).

    CAS  Article  Google Scholar 

  2. Schumacher, M.A. et al. The structural mechanism of GTP stabilized oligomerization and catalytic activation of the Toxoplasma gondii uracil phosphoribosyltransferase. Proc. Natl. Acad. Sci. USA 99, 78–83 (2002).

    CAS  Article  Google Scholar 

  3. Iltzsch, M.H. & Tankersley, K.O. Structure-activity relationship of ligands of uracil phosphoribosyltransferase from Toxoplasma gondii. Biochem. Pharmacol. 48, 781–792 (1994).

    CAS  Article  Google Scholar 

  4. Dubey, J.P. Toxoplasmosis. J. Am. Vet. Med. Assoc. 205, 1593–1598 (1994).

    CAS  PubMed  Google Scholar 

  5. Cleary, M.D., Singh, U., Blader, I.J., Brewer, J.L. & Boothroyd, J.C. Toxoplasma gondii asexual development: identification of developmentally regulated genes and distinct patterns of gene expression. Eukaryot. Cell 1, 329–340 (2002).

    CAS  Article  Google Scholar 

  6. Lekutis, C., Ferguson, D.J., Grigg, M.E., Camps, M. & Boothroyd, J.C. Surface antigens of Toxoplasma gondii: variations on a theme. Int. J. Parasitol. 31, 1285–1292 (2001).

    CAS  Article  Google Scholar 

  7. Aliberti, J. et al. Molecular mimicry of a CCR5 binding-domain in the microbial activation of dendritic cells. Nat. Immunol. 4, 485–490 (2003).

    CAS  Article  Google Scholar 

  8. Raghavan, A. et al. Genome-wide analysis of mRNA decay in resting and activated primary human T lymphocytes. Nucleic Acids Res. 30, 5529–5538 (2002).

    CAS  Article  Google Scholar 

  9. Wang, Y. et al. Precision and functional specificity in mRNA decay. Proc. Natl. Acad. Sci. USA 99, 5860–5865 (2002).

    CAS  Article  Google Scholar 

  10. Ross, J. mRNA stability in mammalian cells. Microbiol. Rev. 59, 423–450 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Yu, Y.T. & Steitz, J.A. Site-specific crosslinking of mammalian U11 and u6atac to the 5′ splice site of an AT-AC intron. Proc. Natl. Acad. Sci. USA 94, 6030–6035 (1997).

    CAS  Article  Google Scholar 

  12. Singh, U., Brewer, J.L. & Boothroyd, J.C. Genetic analysis of tachyzoite to bradyzoite differentiation mutants in Toxoplasma gondii reveals a hierarchy of gene induction. Mol. Microbiol. 44, 721–733 (2002).

    CAS  Article  Google Scholar 

  13. Sibley, L.D., LeBlanc, A.J., Pfefferkorn, E.R. & Boothroyd, J.C. Generation of a restriction fragment length polymorphism linkage map for Toxoplasma gondii. Genetics 132, 1003–1015 (1992).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Bohne, W. et al. Targeted disruption of the bradyzoite-specific gene BAG1 does not prevent tissue cyst formation in Toxoplasma gondii. Mol. Biochem. Parasitol. 92, 291–301 (1998).

    CAS  Article  Google Scholar 

  15. Carninci, P. et al. Normalization and subtraction of cap-trapper-selected cDNAs to prepare full-length cDNA libraries for rapid discovery of new genes. Genome Res. 10, 1617–1630 (2000).

    CAS  Article  Google Scholar 

  16. Ajioka, J.W. et al. Gene discovery by EST sequencing in Toxoplasma gondii reveals sequences restricted to the Apicomplexa. Genome Res. 8, 18–28 (1998).

    CAS  Article  Google Scholar 

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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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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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