BioProject

Poly(A)-tail length and mRNA deadenylation play important roles in post-transcriptional gene regulation. More...

Poly(A)-tail length and mRNA deadenylation play important roles in post-transcriptional gene regulation. Here, we report the regulation of mRNA expression, half-life, and polysome association in mouse embryonic stem cells by the Cnot3 subunit in the Ccr4-Not mRNA deadenylase complex. Overall design: To determine the consequence of Cnot3 deletion, Cnot3 conditional knockout ESCs were first treated with DMSO (WT) or 0.1μM 4-hydroxytamoxifen (4OHT, KO). 1. In one experiment, cells were collected 3 days after the 4OHT treatment and total RNAs were extracted and sequenced to determine changes in mRNA expression. 2. In the second experiment, cells were collected 3 days after the 4OHT treatment and polysome-associated RNAs were fractionated using a published protocol, purified, and sequenced to determine changes in polysome-association. 3. In the third experiment, cells were further treated with actinomycin D (10ug/ml) 2 days after the initial 4OHT treatment. Cells were collected at 0, 4, 8 hrs after actinomycin D treatment and total RNAs were extracted and sequenced to determine changes in mRNA half-life. Two biological replicates were carried out for each experiment. RNAs were extracted using RNA purification kits from Life Technologies, and RNA-seq libraries were prepared using Illumina TruSeq RNA library preparation kit. Less...