Fig. 3: NOT box domain is essential for CNOT3 function in AML.
From: Translation efficiency driven by CNOT3 subunit of the CCR4-NOT complex promotes leukemogenesis
A Experimental scheme of CRISPR/Cas9 tilling sgRNA mutagenesis screen for essential domains of CNOT3 in MOLM13 myeloid leukemia cells. B Top- Graphs presenting z score of individual sgRNAs and smoothed out the distribution of score for functional domains. Read counts for drop-out and/or enrichment of each sgRNA were used to calculate functional scores of gRNAs to specific protein domains using the CRISPRO pipeline. LOESS regression was used to smooth the tiling signals from individual sgRNAs (gray dots) to identify essential amino acids and domains. Bottom–Annotations of CNOT3 structural domains. C Immunoblots showing expression of full-length CNOT3 (CNOT3-FL) and NOT box-truncated proteins. ACTIN serves as loading control. D Cell proliferation of MOLM13 cells transduced with empty vector (EV) control, cDNA expressing full-length CNOT3 or NOT box-truncated CNOT3. Data shown as mean ± s.e.m., n = 3 independent experiments, p < 0.001, two-tailed Student’s t test. E, F Volcano plots showing the distribution of differentially expressed genes upon overexpression of E Full-length CNOT3 (CNOT3-FL) and (F) NOT box-truncated CNOT3 in comparison to empty vector control. G–J Gene set enrichment analysis (GSEA) of RNA-seq dataset shown in E. Source data are provided as Source Data files for figures (B–D).
