Fig. 4

LINC01852 promotes TRIM72-mediated SRSF5 ubiquitination and degradation by interacting with SRSF5 and TRIM72 in CRC cells. (A) RNA pull-down followed by silver-staining was performed to determine differential bands for mass spectrometry analyses to identify LINC01852-associated proteins in CRC cells. (B) RIP assays showed that SRSF5 and TRIM72 enriched LINC01852 in HCT116 cells. (C) Identification of the regions of LINC01852 that mediate the interactions of LINC01852 with SRSF5 and TRIM72. The secondary structure of LINC01852 was predicted via the AnnoLnc2 database. RNA pull-down assays were performed using truncated LINC01852 fragments designed according to the predicted secondary structure. The red area represents the sequence in LINC01852 that binds to SRSF5, and the purple area represents the sequence in LINC01852 that binds to TRIM72. (D) RIP assays were performed to identify the regions in SRSF5 and TRIM72 that mediates their interactions with LINC01852 in HCT116 cells transfected with wild-type or truncation mutants of SRSF5 and TRIM72. (E) The effects of LINC01852 on the protein levels of SRSF5 and TRIM72 were evaluated by WB. (F) Effect of LINC01852 on the endogenous protein level of SRSF5 in CRC cells treated with CHX (50 µg/mL). (G) SRSF5 expression in LINC01852-overexpressing and LINC01852-silenced CRC cells. The cells were treated with MG132 (20 µM) for 6 h before harvest. (H) Effect of LINC01852 on the ubiquitination of SRSF5. HEK-293T cells were transfected with the indicated vectors for 48 h and subjected to ubiquitination assays. Prior to lysis, the cells were treated with MG132 for 6 h. (I) Effect of TRIM72 on the protein level of SRSF5 in CRC cells was detected by WB. (J) Co-IP assays were performed to evaluate the effect of LINC01852 on the interaction between SRSF5 and TRIM72 in HEK-293T cells. The cells were treated with MG132 (20 µM) for 6 h before harvest