RNA analysis by biosynthetic tagging using 4-thiouracil and uracil phosphoribosyltransferase.
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.
Zeiner
Gusti M
GM
Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, USA.
Cleary
Michael D
MD
Fouts
Ashley E
AE
Meiring
Christopher D
CD
Mocarski
Edward S
ES
Boothroyd
John C
JC
D000818:Animals; D019921:Biotinylation; D015152:Blotting, Northern; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D008967:Molecular Biology / Q000379:methods; D010430:Pentosyltransferases / Q000378:metabolism*; D012313:RNA / Q000032:analysis* / Q000096:biosynthesis* / Q000235:genetics; D013379:Substrate Specificity; D013873:Thionucleotides / Q000032:analysis / Q000096:biosynthesis; D013889:Thiouracil / Q000031:analogs & derivatives* / Q000378:metabolism; D014122:Toxoplasma / Q000201:enzymology
D013873:Thionucleotides; C114719:4-thiouracil; D013889:Thiouracil; D012313:RNA; D010430:Pentosyltransferases; C023269:uracil phosphoribosyltransferase