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. Author manuscript; available in PMC: 2021 Aug 15.
Published in final edited form as: Cancer Res. 2021 Feb 15;81(4):1087–1100. doi: 10.1158/0008-5472.CAN-20-1807

Figure 4: JMJD6 regulates AR-V7 transcription, in part, through recruitment of splicing factor U2AF65 to AR-V7 specific splice sites in in vitro models of CRPC.

Figure 4:

(A-C) Scatter plots showing correlations between JMJD6 mRNA expression and (A) androgen response (Hallmark; H), (B) AR signature (derived from 43 AR regulated transcripts) and (C) AR-V7 signature (derived from 59 genes associated with AR-V7 expression in mCRPC) in 159 mCRPC biopsies (SU2C/PCF cohort). U2AF65 mRNA expression shown as log FPKM. r-values and p-values are shown and were calculated using Spearman’s correlation. (D) Single WB in technical triplicate demonstrating reduction in AR-V7 protein levels with both JMJD6 and U2AF65 siRNA in 22Rv1 PC cells. JMJD6 siRNA had minimal impact on U2AF65 protein levels. (E) Schematic diagram of the human AR gene illustrating the regions targeted in RNA immunoprecipitation (RIP) assay with accompanying summary bar chart. Shows a reduction in detectable U2AF65 at the AR-V7 specific splice sites P1 (containing the 5’ splice site for both AR and AR-V7) and P2 (containing the 3’ splice site for AR-V7) in 22Rv1 PC cells treated with JMJD6 siRNA compared to non-targeting control siRNA. Indicates that JMJD6 regulates recruitment of the splicing factor U2AF65 to AR-V7 splice sites. RIP data derived from two independent experiments conducted in triplicate. p values (*, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001) were calculated for each condition compared to control (at equivalent concentration) using the mean value of technical replicates with unpaired Student’s t tests. (F) Schematic representation of alternative splicing events alongside corresponding histogram of alternative splicing mean differences between non-targeting control siRNA (blue dotted line; defined as 0.0) and JMJD6 siRNA in LNCaP95 PC cells. Left shift denotes decrease in splicing events. Total number of alternative splicing events (x) occurring in total number of genes (y) shown in orange (x/y). JMJD6 knockdown led to substantial changes in 753 alternative splicing events, with the majority of these occurring less frequently. (G) JMJD6 knockdown in LNCaP95 PC cells associated with a reduction in AR-V7 activity (derived from 59 genes associated with AR-V7 expression in mCRPC); Enrichment Score (ES) = −0.32.