Exploring RNA transcription and turnover in vivo by using click chemistry.
We describe a chemical method to detect RNA synthesis in cells, based on the biosynthetic incorporation of the uridine analog 5-ethynyluridine (EU) into newly transcribed RNA, on average once every 35 uridine residues in total RNA. EU-labeled cellular RNA is detected quickly and with high sensitivity by using a copper (I)-catalyzed cycloaddition reaction (often referred to as "click" chemistry) with fluorescent azides, followed by microscopic imaging. We demonstrate the use of this method in cultured cells, in which we examine the turnover of bulk RNA after EU pulses of varying lengths. We also use EU to assay transcription rates of various tissues in whole animals, both on sections and by whole-mount staining. We find that total transcription rates vary greatly among different tissues and among different cell types within organs.
Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA.
Cindy Y
CY
Jao
Adrian
A
Salic
D000825:Animal Structures / Q000378:metabolism; D000818:Animals; D001386:Azides; D002478:Cells, Cultured; D019411:Clinical Laboratory Techniques; D005456:Fluorescent Dyes; D007700:Kinetics; D008853:Microscopy; D012313:RNA / Q000032:analysis / Q000096:biosynthesis*; D014158:Transcription, Genetic; D014498:Uracil / Q000031:analogs & derivatives; D014529:Uridine / Q000031:analogs & derivatives* / Q000378:metabolism
D001386:Azides; D005456:Fluorescent Dyes; C073482:eniluracil; D014498:Uracil; D012313:RNA; D014529:Uridine