Fig. 4

SUMOylation of KHSRP at K87 inhibits the biogenesis of miRNAs. (A-B) The mutation K87R of KHSRP increases its interaction with Drosha and DGCR8. 293T cells were transfected with HA-Drosha and Flag-KHSRP-WT or Flag-KHSRP-K87R (a), or transfected with Flag-DGCR8 and HA-KHSRP-WT or HA-KHSRP -K87R (b). 48 h after transfection, cells were lysed for immunoprecipitation with anti-Flag antibody, and followed by Western blotting with indicated antibodies. The band intensities were calculated by ImageJ software and the ratios were quantified. c KHSRP SUMOylation regulates the biogenesis of a subset of miRNAs. High-throughput deep sequencing data showed that there were 368 miRNAs whose RPM (reads per million) > 10 RPM in all expressed miRNAs of DU145 control cell line. 151 of these miRNAs whose expression in DU145-shKHSRP cells were downregulated compared to those in DU145-shRNA control cells. 51 of 151 miRNAs whose expression were upregulated in DU145 shKHSRP-KHSRP-K87R cells compared to those in DU145 shKHSRP-KHSRP-WT cells. d KHSRP-K87R promotes some miRNAs biogenesis. According to sequencing data, a total of 51 miRNAs whose expression were upregulated in DU145 shKHSRP-KHSRP-K87R cells compared to those in DU145 shKHSRP-KHSRP-WT cells. Among 51 miRNAs, there were only 6 miRNAs which harbor only one single G or none G (labeled with *). Detailed can be seen Additional file 10: Table S4. e The biogenesis of some miRNAs promoted by KHSRP-K87R was validated by qRT-PCR. qRT-PCR was performed to assess the expression of endogenous let-7i-5p, miR-98-5p, miR-182a-5p and miR-183a-5p in DU145 shKHSRP-KHSRP-WT and shKHSRP-KHSRP-K87R stable cell lines