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

本研究采用mPAT方法，使用锚定寡聚dT（anchored oligo-dT）引物TV12VN，以探究RNA聚合酶II（RNA polymerase II）慢突变体rpb1-1酵母的3'非翻译区（3' untranslated region, 3'UTR）长度变化情况；该酵母菌株分别互补携带野生型RPB1或慢突变rpb1质粒，并经虫草素处理，同时通过4-硫尿嘧啶（4-thiouracil, 4tU）标记RNA以追踪新转录过程，测序读段中呈现T到C的碱基转换特征。整体实验设计：将携带野生型RPB1质粒pCK859或rpb1-H1085Y慢突变质粒pCK870的rpb1-1酵母细胞在25℃下培养至OD600为0.6，随后加入20μg/ml虫草素与500μM 4tU，或等体积的二甲基亚砜（DMSO），分别处理0、20、40分钟后收获细胞样本。最终采用Illumina MiSeq测序平台，通过锚定寡聚dT引物的mPAT方法完成样本分析。