PT - JOURNAL ARTICLE AU - Heather J. Painter AU - Manuela Carrasquilla AU - Manuel LlinĂ¡s TI - Capturing <em>in vivo</em> RNA transcriptional dynamics from the malaria parasite <em>P. falciparum</em> AID - 10.1101/099549 DP - 2017 Jan 01 TA - bioRxiv PG - 099549 4099 - http://biorxiv.org/content/early/2017/01/12/099549.short 4100 - http://biorxiv.org/content/early/2017/01/12/099549.full AB - To capture the transcriptional dynamics within proliferating cells, methods to differentiate nascent transcription from pre-existing mRNAs are desired. One approach is to label newly synthesized mRNA transcripts in vivo through the incorporation of modified pyrimidines. However, the human malaria parasite, Plasmodium falciparum, is incapable of pyrimidine salvage for mRNA biogenesis. To capture cellular mRNA dynamics during Plasmodium development, we have engineered parasites that can salvage pyrimidines through the expression of a single bifunctional yeast fusion gene, cytosine deaminase/uracil phosphoribosyltransferase (FCU). We show that expression of FCU allows for the direct incorporation of thiol-modified pyrimidines into nascent mRNAs. Using developmental stage-specific promoters to express FCU-GFP enables the biosynthetic capture and in-depth analysis of mRNA dynamics from subpopulations of cells undergoing differentiation. We demonstrate the utility of this method by examining the transcriptional dynamics of the sexual gametocyte stage transition, a process that is essential to malaria transmission between hosts. We find that sexual stage commitment is governed by transcriptional reprogramming and the stabilization of a subset of essential gametocyte transcripts. This new method for biosynthetic labeling of Plasmodium mRNAs is incredibly versatile and can be used to measure transcriptional dynamics at any stage of parasite development, and thiol-modified RNAs will allow for future applications to measure RNA-protein interactions in the malaria parasite.