Cnot1 preferentially interacts with anti-proliferative gene transcripts in a Cnot3-dependent manner. (a) Cnot1 interaction with mRNAs. RIP was performed in hESC-derived cardiomyocytes with either IgG or Cnot1 antibody. Relative abundance of the immunoprecipitated mRNAs was determined by RT-qPCR, normalized to Input, and plotted as mean ± SEM from three independent experiments. (b) Effects of Cnot3 silencing on Cnot1 RIP. hESC-derived cardiomyocytes were transduced with shCnot3, and RIP was conducted 48 h after transduction. Values were plotted as mean ± SEM from three independent experiments. (c) Cnot1 RIP sequencing. Cnot1 RIP was performed in hESC-derived cardiomyocytes, and the total and Cnot1-interacting RNAs were subjected to high-throughput sequencing. Representative images form the genome browser were shown for the indicated genes. For each gene, upper track: Cnot1 RIP; lower track: Input. (d) Statistical analysis of Cnot1 RIP-sequencing. Genes detected in the sequencing were ranked by their expression level in hESC-derived cardiomyocytes and divided into four equal groups (Q1–Q4). Cnot1 binding was determined by the ratio of the reads per kilo base per million (RPKM) in Cnot1 RIP/Input and compared between Proliferative and Anti-proliferative genes in each quarter. Proliferative genes were defined as those downregulated in long-term cultured hESC-CMs, while Anti-proliferative genes were defined as those upregulated (See Experimental Procedures for details). P values were calculated by the Wilcoxon rank-sum test.