Figure 1.
(A) GAS5 mRNA was detected by RT-PCR after RNA-IP using eIF4E and IgG antibodies. The RNA-IP was repeated for two times with similar results. (B) The polysome and non-polysome fractions, as shown by the profile of the absorbance at 254 nm, were separated by sucrose gradient centrifugation in HEK-293T cells. The experiments were repeated three times. (C) The abundance of GAS5 lncRNA in the polysome and non-polysome fractions was measured by Q-PCR and normalized with total GAS5 mRNA in all the fractions. Bars represent mean ±SD from 3 replicates. Experiment was repeated three times, and a representative experiment is shown. (D) Protein levels of eIF4E and eIF4G in the polysome and non-polysome fractions were detected by western blot.
Figure 2.
GAS5 binds to eIF4E through RNA binding motifs.
(A) RNA binding motifs, which are italic and bold, in the eIF4E protein were predicted with 2 web-based tools, BindN and PPRInt. The motif, W56, W102 and E103 for m7G binding is bold and underlined. N-terminally located sequence is motif-1 and the C-terminal one is motif-2. (B-C) GAS5 RNA was detected by RT-PCR after RNA-IP assay using HA antibody in the cells transfected with (B) RNA binding deletion mutants (HA-eIF4EDel1, HA-eIF4EDel2 and HA-eIF4EDel1&2) and (C) cap binding mutant (HA-eIF4Ecap mutant). GAPDH was used as a control.
Figure 3.
GAS5 suppresses c-Myc expression at protein level.
(A) GAS5 was knocked down by siRNA in HEK-293T cells, and global protein translation was measured by [3H]-leucine incorporation assay. Bars represent mean ±SD from 3 replicates for Q-PCR and 6 replicates for [3H]-leucine incorporation assay. The experiments were performed two times. (B) pRF and pRF-SL plasmid were transfected into HEK-293T cells and cap-dependent protein translation efficiency was evaluated by luciferase assay. Bars represent mean ±SD from 4 replicates. The data were repeated three times. (C) The protein level of c-Myc (P = 0.007), Mcl1 (P = 0.88), survivin (P = 0.47) and Bcl2 (P = 0.47) was assessed by western blot (n = 3) after the cells were treated with GAS5 or control siRNA. (D) The mRNA level of c-Myc (P = 0.10), Mcl1 (P = 0.75), survivin (P = 0.31) and Bcl2 (P = 0.19) was quantified by Q-PCR after the cells were treated with GAS5 or control siRNA. Bars represent mean ±SD from 3 replicates. The experiment was repeated three times. (E) The protein level of c-Myc (P = 0.005) was assessed by western blot (n = 3) after the cells were transfected with in vitro transcribed GAS5 RNA. (F-G) The mRNA level of c-Myc (P = 0.37) (F) and GAS5 (G) was quantified by Q-PCR after the cells were transfected with in vitro transcribed GAS5 RNA. Bars represent mean ±SD from 3 replicates. The experiment was performed three times.
Figure 4.
GAS5 suppresses c-Myc translation through direct binding with its mRNA.
(A) The protein level of c-Myc was assessed by western blot after the cells were treated with GAS5 or control siRNA followed by the treatment of 100 µg/ml CHX for 1 and 2 hours. (B) c-Myc mRNA level associated with the polysome and non-polysome fractions of GAS5 and control siRNA transfected HEK-293T cells were evaluated by Q-PCR. Data was normalized with total mRNA (non-polysome + polysome). Bars represent mean ±SD from 3 replicates. (C) RNA from HEK-293T cells was pulled down by biotin-labeled GAS5 antisense DNA oligo with GAS5 sense DNA oligo as a control. The binding of c-Myc mRNA to GAS5 was evaluated by RT-PCR, in which GAPDH was used as a control.
Figure 5.
GAS5 cooperates with eIF4E to regulate c-Myc translation.
(A-B) eIF4E expression at mRNA (A) and protein level (B) was assessed by Q-PCR and western blot after the cells were treated with GAS5 or control siRNA. Bars represent mean ±SD from 3 replicates. The experiment was performed three times. (C) GAS5 expression was quantified by Q-PCR in the HEK-293T cells transfected with non-silencing or eIF4E shRNA. Bars represent mean ±SD from 3 replicates. (D). eIF4E protein level after the cells were stably transfected with non-silencing or eIF4E shRNA. (E) The protein level of c-Myc and eIF4E was assessed after the cells were transfected with GAS5/control siRNA and eIF4E plasmid.
Figure 6.
A schematic diagram of the GAS5 and c-Myc translation regulation.