Pyrimidine salvaging enzyme UPRTase is active in Drosophila and limits the specificity of tissue specific RNA isolation by 4TU tagging. Arpan Ghosh, MaryJane Shimmel, Emma Leof, Michael O'Connor. Gen Cell & Development, Univ Minnesota Twin Cities, Minneapolis, MN.

   Spatial-temporal regulation of gene expression is central to the existence of multicellular organisms. However, studying cell/tissue specific gene expression is often limited by the ability to isolate homogeneous populations of specfic cell types. Recently T. gondii Uracil-phophorybosyltransferase (Tg-UPRT) mediated 4-thiouracil (4TU) tagging has been described as an efficient method for tissue/cell-type specific RNA isolation (Miller, MR et. al. Nature methods 2009). Specificity and efficiency of this technique is based on the present understanding that all UPRT homologues from higher eukaryotes are inactive. Here we show that Drosophila UPRT homologue CG5537 (krishah, kri) is active in vivo and is essential for larval growth. Both S2 cells and larvae are capable of efficiently incorporating 4TU, and kri-RNAi can significantly reduce this incorporation. Additionally, loss of kri severely affects larval growth and gives rise to thin larvae that, in some cases, form thin pupae/pre-pupae that mostly die before reaching the pharate stage. However, developmental timing of stage transitions in the mutant larvae is not affected. Interestingly, we show that a relatively weaker kri-RNAi can significantly reduce 4TU incorporation without causing larval growth defects. This provides the possibility of systemically knocking down kri to reduce background incorporation of 4TU while using Tg-UPRT to obtain tissue specific RNA tagging. Towards this goal we have verified that the kri-RNAi does not affect expression of a codon-optimized Tg-UPRT construct or its ability to incorporate 4TU. Overall we provide strong evidence to show that, contrary to current understanding, the Drosophila UPRT homologue kri, is active in vivo. We also suggest ways of significantly improving the specificity and efficiency of a promising cell-type/tissue specific RNA isolation technique.