The cancer-associated RNA-binding protein La is posttranslationally modified by phosphorylation and sumoylation. STAT3 restored cell proliferation partially. Finally, we present RNA sequencing data from RNA immunoprecipitations (RIPs) and record that mRNAs from the cell routine and ubiquitination are preferentially destined by GFP-LaWT and are less enriched in GFP-LaSD RIPs. Taken together, results of our study support a novel mechanism whereby sumoylation of La promotes cell proliferation by averting ubiquitination-mediated degradation of the STAT3 protein. = 3). (B) RT-PCR on RNA samples prepared from RIP experiments using HEK293 cells stably overexpressing GFP-LaWT (Wt) or GFP-LaSD (K41/200R). Significantly less STAT3 mRNA was associated with GFP-LaSD than with GFP-LaWT. The asterisks indicate a significant difference ( 0.01), as determined by Student’s test (= 3). The data represent means and SD of the results of independent experiments. Open in a separate window FIG 6 Sumoylation of La promotes cell proliferation via a STAT3-mediated mechanism. (A) Representative immunoblot of STAT3 in HEK293 cells transduced with shC, sh3, and sh5 lentiviral constructs. GAPDH was used as a loading control. (B) RT-qPCR analysis showing no significant difference Anacetrapib (MK-0859) in STAT3 mRNA levels in HEK293 cells transduced with shC, sh3, and sh5 lentiviral constructs. The values are normalized against GAPDH mRNA levels (= 3). (C) Representative immunoblot showing STAT3 protein levels in GFP-LaWT- and GFP-LaSD-expressing cells. GAPDH was used as a loading control. (D) Densitometry analysis showing significantly lower STAT3 protein expression in GFP-LaSD-expressing cells than in GFP-LaWT-expressing cells. The values are normalized against GAPDH protein levels (= 3). (E) Representative fluorescence images showing transient transfection of control (GFP) and RFP-STAT3 in cells transduced with lentiviral constructs expressing shC or sh5. The transfection efficiency was 30%. (F) Overexpression of STAT3 (RFP-STAT3) restored the numbers of La-depleted (sh5) cells (= 4; *, = 0.0358). (G) Representative fluorescence images showing transient transfection of control and RFP-STAT3 in GFP-, GFP-LaWT-, and GFP-LaSD-expressing cells. The transfection efficiency was 30%. (H) Overexpression of STAT3 (RFP-STAT3) restored GFP-LaSD cell numbers (= 3; *, = 0.015). The asterisks indicate significant differences ( 0.05), as determined by Student’s test. NS, not significant. The data represent means and SD of the results of independent experiments. To test whether the GFP-LaWT bands represented a sumoylated La isoform, we immunoprecipitated GFP-LaWT and GFP-LaSD using cell lysates prepared from HEK293 cells STAT6 stably overexpressing GFP-LaWT, GFP-LaSD, or GFP (Fig. 2A; see Fig. 6C). As we hadseen for the immunoprecipitation of sumoylated endogenous La (Fig. 1D), we found that the immunoprecipitation of the GFP-tagged high-molecular-mass La isoforms is very inefficient (Fig. 3A to ?toC).C). In the case of GFP-La immunoprecipitation, we did not pull down the high-molecular-mass bands 1 to 4, as seen in the immunoblot (Fig. 2B and ?and3A);3A); however, we detected a La-specific band (Fig. 3B, arrow), which was also recognized by a GFP-specific antibody (Fig. 3C, arrow). This band was also detected with the SUMO2/SUMO3-specific (Fig. 3D, arrow) and SUMO1-specific (Fig. Anacetrapib (MK-0859) 3E, arrow) antibodies. Note that we have recently reported that recombinant La can be sumoylated by SUMO1, SUMO2, and SUMO3 (42). The intensity of the bands recognized by the SUMO2/SUMO3-specific antibody was weaker in immunoprecipitations from GFP-LaSD than in the GFP-LaWT immunoprecipitations (Fig. 3D and ?andE).E). The band is very similar in size to that of endogenous sumoylated La; however, the band can be identified by the GFP-specific antibody also, recommending it represents a sumoylated cleavage product of GFP-La strongly. We tried many solutions to stabilize customized GFP-La during cell lysis (e.g., lysis in popular SDS or 5 M urea), without success unfortunately. Altogether, we offer proof that endogenous, aswell as GFP-tagged, La can be sumoylated in HEK293 cells. The type of the excess music group detectable by immunoblotting from GFP-LaSD cells (Fig. 3A, hash tag) isn’t clear, as well as the music group is not often obviously detectable (Fig. 2B) (42). This music group shows up when two Anacetrapib (MK-0859) lysine residues are mutated, that could result in the usage of substitute sites for sumoylation of La, and extra sumoylation sites have already Anacetrapib (MK-0859) been reported.