Effect of depleting GNB1L on PIKK mRNA levels to explore the regulatory mechanism of GNB1L on PIKK proteins
Our screens identified GNB1L, a high-confidence hit, as a novel positive regulator of DDR signaling. We therefore generated HEK293A-GNB1L-dTAG cells which the GNB1L-dTAG-HA protein decreased to markedly lower levels in the presence of dTAGv-1 than that in cells treated with dTAG-NEG , we next examined whether depletion of GNB1L affects DDR signaling. Notably, depletion of GNB1L diminished all IR-induced DDR signaling. Moreover, depletion of GNB1L also significantly reduced basal DDR signaling as well as PIKKs. To determine whether GNB1L depletion specifically affects PIKKs instead of other DDR factors, we carried out label-free quantitative proteomics analysis by comparing the proteomes in NEG-treated versus in dTAGv-1-treated GNB1L-dTAG cells. The data showed that GNB1L depletion specifically reduced PIKK kinases and ATRIP rather than other DDR factors. Obviously, PIKK kinases, ATRIP and mTOR are major downregulated substrates affected by GNB1L depletion. Therefore, we performed RNA-seq to rule out the possibility that GNB1L regulates PIKK protein levels via transcriptional regulation. The differentially expressed genes (DEGs) as well as the GO analysis confirmed that depleting GNB1L had no effect on PIKK mRNA levels . These data suggested that GNB1L specifically regulates PIKK protein levels instead of other DDR factors. Overall design: To investigate whether depletion of GNB1L affects PIKK mRNA levels therefore influencing PIKK proteins, we generated HEK293A-GNB1L-dTAG cells which the GNB1L-dTAG-HA protein decreased to markedly lower levels in the presence of dTAGv-1 than that in cells treated with dTAG-NEG. HEK293A-GNB1L-dTAG cells were treated with 1 µM NEG or dTAGv-1 for 3 days followed by RNA extraction with kit (Qiagen; 217004). We prepared 3 replicates for each group. All the following mRNA sequencing processes until the generation of FASTQ files were completed by Cancer Genomics Center at the University of Texas Health Science Center at Houston. Total RNA was extracted from each condtion and RNAseq was performed and analysed for the differentially expressed genes (DEGs) as well as the GO analysis.