Comprehensive analysis of CCR4-NOT complex-mediated RNA regulation with inducible-gene knockout cells of CCR4-NOT complex subunits
CCR4-NOT complex is a multi-subunit protein complex, which executes the degradation of poly(A) tails of mRNA and is also involved in transcription and translation both directly and indirectly. We had identified CCR4-NOT complex as a crucial regulator of heart function and found Atg7-mediated cardiomyocyte damage and heart failure in CCR4-NOT-depleted mice (Cell 2010, Sci Signaling 2018). To determine precise roles of CCR4-NOT complex in RNA regulation, we generated tamoxifen (4OHT) inducible-gene knockout cells (mouse embryonic fibroblasts) of CCR4-NOT complex subunits (Cnot1, Cnot3, or Cnot7/8) and analyzed the acute effects of CCR4-NOT depletions on global gene expression. For this purpose, we conducted RNA-seq for transcriptome, 4sU-labeling and sequencing for transcription, sequencing after Actinomycin D treatment for mRNA stability, and ribosome profiling (RIBO-seq) for translation in these CCR4-NOT-depleted cells. In addition, we also conducted Cnot1 RIP-seq to isolate target mRNAs of CCR4-NOT complex.