Supplementary Figure 3: AES Interacts with RNA Helicase DDX21 and Multiple SnoRNAs.
From: AML1-ETO requires enhanced C/D box snoRNA/RNP formation to induce self-renewal and leukaemia
(a) Schematic diagram of nascent RNA-Seq. Control (shCtr) and AES knockdown (shAES) Kasumi-1 cells (two replicates for each) were cultured in medium containing 4-thiouridine for 1 h. 4-thiouridine was metabolically incorporated into newly transcribed (nascent) RNA during transcription. Following isolation of total cellular RNA and thiol-specific biotinylation, nascent RNA was separated from total RNA using streptavidin-coated magnetic beads. Paired-end libraries prepared from nascent RNA were sequenced on an Illumina Hiseq2000 platform. (b) Levels of Nascent transcripts (FPKM) of snoRNA host genes in control (shCtr) and AES knockdown cells (shAES). 36 host genes for snoRNAs that are downregulated by AES knockdown were shown. (c) Visualization nascent RNA-seq reads on snoRNA host gene RPL3. (d,e) Gene ontology analysis of downregulated nascent transcripts upon AES knockdown. 2180 transcripts were found to be downregulated in AES knockdown Kasumi-1 cells (FPKMshAES/FPKMshCtr < 0.7 and FPKMshCtr > 1). (f) AES and DDX21 interact in vivo. 293T cells were transfected with V5-tagged AES. Immunoprecipitation was performed with anti-V5 antibody. Western blot analysis was performed with anti-DDX21 or anti-V5 antibody. (g) Western blot indicates DDX21 expression in Kasumi-1 leukemic cells with DDX21 knockdown by shRNA, compared to control shRNA (against luciferase gene, shLUC). (h) RT-PCR analysis of snoRNAs in control and DDX21 knockdown Kasumi-1 cells. Data are represented as mean ± s.d. of n = 3 independent experiments. (i) Kasumi-1 cells were infected with lentivirus expressing control shRNA (shLUC), shRNA against AES or DDX21. Percentage of infected cells (GFP+) was analyzed at the indicated time points. Percentage was normalized to the number at day 1. Error Bar, mean ± s.d. n = 3 independent experiments. (j) Cell cycle analysis of control (shLUC) Kasumi-1 cells and Kasumi-1 knockdown of AES (shAES), DDX21 (shDDX21). Error Bar, mean ± s.d. n = 3 independent experiments. Unprocessed original scans of blots are shown in Supplementary Figure 7. Statistical source data for Supplementary Fig. 3j are provided in Supplementary Table 7.
