Genetic and pharmacological evidence for kinetic competition between alternative poly(A) sites in yeast - 4tU cordycepin treatment with rpb1 slow mutant

mPAT approach using an anchored oligo-dT (TV12VN) primer to determine changes to 3'UTR length in yeast with a slow RNA polymerase II mutant rpb1-1, complemented with wild-type RPB1 or a slow rpb1 mutant plasmid, upon cordycepin treatment and new transcription using 4-thiouracil (4tU) labelling of RNA. This appears as a T to C conversion in sequencing reads. Overall design: rpb1-1 yeast cells carrying plasmids with either RPB1 wild-type (pCK859) or the rpb1-H1085Y slow mutation (pCK870) were grown at 25ºC until an OD600 of 0.6 was reached then 20µg/ml cordycepin and 500µM 4tU or equivalent volumes of DMSO added for 0, 20 or 40 minutes before harvesting. Samples were analysed via mPAT with an anchored oligo-dT primer using the Illumina MiSeq platform.