RNA analysis by biosynthetic tagging using 4-thiouracil and uracil phosphoribosyltransferase

• Published in: Methods in molecular biology (Clifton, N.J.) Vol. 419; p. 135
• Main Authors: Zeiner, Gusti M, Cleary, Michael D, Fouts, Ashley E, Meiring, Christopher D, Mocarski, Edward S, Boothroyd, John C
• Format: Journal Article
• Language: English
• Published: United States 2008
• Subjects: Animals; Biotinylation; Blotting, Northern; Gene Expression Profiling; Gene Expression Regulation; Molecular Biology - methods; Pentosyltransferases - metabolism; RNA - analysis; RNA - biosynthesis; RNA - genetics; Substrate Specificity; Thionucleotides - analysis; Thionucleotides - biosynthesis; Thiouracil - analogs & derivatives; Thiouracil - metabolism; Toxoplasma - enzymology
• ISSN: 1064-3745
• DOI: 10.1007/978-1-59745-033-1_9

RNA analysis by biosynthetic tagging (RABT) enables sensitive and specific queries of (a) how gene expression is regulated on a genome-wide scale and (b) transcriptional profiling of a single cell or tissue type in vivo. RABT can be achieved by exploiting unique properties of Toxoplasma gondii uracil phosphoribosyltransferase (TgUPRT), a pyrimidine salvage enzyme that couples ribose-5-phosphate to the N1 nitrogen of uracil to yield uridine monophosphate (UMP). When 4-thiouracil is provided as a TgUPRT substrate, the resultant product is 4-thiouridine monophosphate which can, ultimately, be incorporated into RNA. Thio-substituted nucleotides are not a natural component of nucleic acids and are readily tagged, detected, and purified with commercially available reagents. Thus, one can do pulse/chase experiments to measure synthesis and decay rates and/or use cell-specific expression of the TgUPRT to tag only RNA synthesized in a given cell type. This chapter updates the original RABT protocol (1) and addresses methodological details associated with RABT that were beyond the scope or space allotment of the initial report.