SMaRT lncRNA controls translation of a G-quadruplex containing mRNA antagonizing the DHX36 helicase (2020)
Figure 3

Figure 3 - Lnc-SMaRT molecular interactome.

A Localization on the lnc-SMaRT transcript of the two sets of biotinylated probes (Set#1 and Set#2) used for lnc-SMaRT pull-down experiment.

B Left panel: qPCR analysis of lnc-SMaRT enrichment in the RNA pull-down performed in C2C12 cells at day 2 of differentiation (DM2); Set#1 and Set#2 probes were used against lnc-SMaRT together with a control set of probes against LacZ mRNA (LacZ). Data are expressed in percentage of input and presented as the mean ± s.e.m. of three biological replicates (dots).

Right panel: Western blot analysis showing the specific enrichment of the DHX36 helicase in lnc-SMaRT pull-down; GAPDH was used as the negative control. Representative results from three independent experiments are shown.

C Upper panel: Western blot with DHX36 antibodies on protein extracts from DHX36 RNA immunoprecipitation. Input sample (IN) accounts for 2.5% of the extract. Representative results from three independent experiments are shown.

Lower panel: qPCR analysis of lnc-SMaRT enrichment in DHX36 RIP-derived RNA extracts. WBP4 was used as positive control [31], while Neat1 (a lncRNA expressed at comparable level of lnc-SMaRT according to the RNA-sequencing) and Rps7 were used as negative controls. Data are expessed as percentage of input normalized on IgG control, and presented as the mean ± s.e.m. of three biological replicates (dots).

D RT-PCR validation of Mlx-α, Mlx-β and MLX-γ mRNA enrichment upon the lnc-SMaRT pull-down performed with Set#1 and Set#2 probes; a control set of probes against LacZ mRNA (LacZ) was used as negative control. GAPDH was used as negative control. Input sample (IN) accounts for 10% of the extract. Representative results from three independent experiments are shown.

E RT-PCR analysis of Mlx-α, Mlx-β and Mlx-γ mRNA enrichment in DHX36 RIP-derived RNA extracts in samples treated with control siRNA (siSCR, upper panel) or siRNA against lnc-SMaRT (siSMaRT, lower panel). GAPDH was used as negative control. Input sample (IN) accounts for 10% of the extract. Representative results from three independent experiments are shown.

F Upper panel: Western blot with FLAG antibody on protein extracts from HeLa cells overexpressing the indicated isoforms of FLAG-tagged MLX (MLX-α, MLX-β and MLX-γ) in control condition (CTRL) or in overexpression of lncSMaRT (OE-SMaRT). FLAG-tag has been inserted at the N-terminus of MLX protein isoforms. HPRT was used as loading control. Representative results from three independent experiments are shown.

Middle panel: Quantification of FLAG-tagged protein levels normalized on HPRT signals. Data are expressed presented as the mean ± s.d. of three biological replicates (dots). Statistical analysis was performed with two-way ANOVA followed by Bonferroni's multiple comparisons test. ***P < 0.001.

Lower panel: RT-PCR analysis of lnc-SMaRT and Mlx expression on RNA extracts from HeLa cells overexpressing the indicated isoforms of flagged-MLX (MLX-α, MLX-β and MLX-γ) in control condition (CTRL) or in overexpression of lncSMaRT (OE-SMaRT). GAPDH was used as loading control. Representative results from three independent experiments are shown.

G Upper panel: Western blot with FLAG antibodies on protein extracts from N2a cells transfected with an empty vector (CTRL) or with a plasmid overexpressing FLAG-tagged MLX-γ (MLX-γ) in control conditions (si-SCR) or in knockdown of DHX36 (si-DHX36). ACTININ was used as loading control. Representative results from three independent experiments are shown.

Lower panel: RT-PCR analysis of Mlx-γ expression on the described RNA extracts. GAPDH was used as loading control. Representative results from three independent experiments are shown.