7SL RNA represses p53 translation by competing with HuR

Abstract

Noncoding RNAs (ncRNAs) and RNA-binding proteins are potent post-transcriptional regulators of gene expression. The ncRNA 7SL is upregulated in cancer cells, but its impact upon the phenotype of cancer cells is unknown. Here, we present evidence that 7SL forms a partial hybrid with the 3'-untranslated region (UTR) of TP53 mRNA, which encodes the tumor suppressor p53. The interaction of 7SL with TP53 mRNA reduced p53 translation, as determined by analyzing p53 expression levels, nascent p53 translation and TP53 mRNA association with polysomes. Silencing 7SL led to increased binding of HuR to TP53 mRNA, an interaction that led to the promotion of p53 translation and increased p53 abundance. We propose that the competition between 7SL and HuR for binding to TP53 3'UTR contributes to determining the magnitude of p53 translation, in turn affecting p53 levels and the growth-suppressive function of p53. Our findings suggest that targeting 7SL may be effective in the treatment of cancers with reduced p53 levels.

Figure 1.

7SL is highly expressed in cancer tissues and is required for cell growth. (A) The abundance of 7SL in the tumor tissues (T) indicated (liver, lung, breast, stomach) and adjacent normal tissues (N) was measured by RT-qPCR analysis and normalized to 18S rRNA levels. (B) RT-qPCR analysis of 7SL levels 48 h after transfection of HeLa cells with Ctrl siRNA or 7SL siRNA. (C) Forty-eight hours after siRNA transfection of HeLa, Mia PaCa-2, HCT116 or RKO cells, cell numbers were measured using a hemocytometer and represented as % of cells relative to the Ctrl group. (D) Growth kinetics of HeLa cells transfected with Ctrl siRNA or 7SL siRNA. Cells were counted daily for 4 days. Data in (B)–(D) are the means ± S.D. from three independent experiments.

Figure 2.

7SL binds TP53 mRNA and lowers p53 abundance. (A) Sequence alignment of 7SL and TP53 mRNA using BLAST shows potential sense–antisense interactions. (B) TP53 mRNA pulldown using the biotin-ASOs shown (see the Materials and Methods section) was followed by RT-qPCR analysis to quantify TP53 mRNA and 7SL levels; GAPDH mRNA was used for normalization. (C) HeLa cells were transfected with the indicated siRNAs; 48 h later, the levels of p53, p21 and loading control β-actin were assessed by western blot analysis. (D) The levels of TP53 and CDKN1A mRNA (encoding p21) and normalization control 18S rRNA were quantified by RT-qPCR. (E) Forty-eight hours after transfecting plasmids pcDNA3 or pcDNA3-7SL, total cellular 7SL levels were measured by RT-qPCR analysis (left) and the levels of p53 and loading control HSP90 by western blot analysis (right). Data in (B) and (E) are the means + S.D. from three independent experiments. In (C) and (E), the means ± standard deviation of p53 signals are indicated and the p values are shown.

Figure 3.

(A) HeLa cells were transfected with siRNAs directed to SRP proteins and 48 h later the levels of the mRNAs encoding each SRP protein were measured by RT-qPCR (left) and the levels of protein were assessed by western blot analysis (right; the SRP19 antibody was not adequate for this analysis, not shown) and loading was monitored by detecting the housekeeping control protein β-actin. (B) In cells processed as in (A), the levels of TP53 mRNA were measured by RT-qPCR (left) and the levels of p53 protein (as well as loading control β-actin) were assessed by western blot analysis (right). This group included analysis of cells in which SRP68, SRP54 and SRP19 were silenced simultaneously (‘ALL SRP siRNAs’). (C) In cells processed as in (A), the levels of 7SL were measured by RT-qPCR analysis. (D) Forty-eight hours after transfection as in (A), cells were counted using a hemocytometer. In (A)–(D), data are shown as the means + S.D. from three independent experiments. (E)–(G) From each of 12 fractions from glycerol gradients shown in the global RNA profile (10-40% glycerol) (E), RNA was isolated and used for RT-qPCR analysis of 7SL levels (F), and protein was precipitated and used for western blot analysis of SRP54 and SRP68. Data are representative of three independent experiments.

Figure 4.

Competitive regulation of p53 expression by 7SL and HuR. (A) Schematic of TP53 mRNA depicting potential binding sites for 7SL (green) and HuR (yellow). The thick grey lines and numbers above and below TP53 3′UTR depict regions of complementarity with 7SL. (B) HeLa cells were transfected with 7SL siRNA; 48 h later, HuR association with the indicated mRNAs was quantified by RIP analysis. Data were normalized to the levels of GAPDH mRNA in each IP sample and represented as the enrichment of each mRNA relative to the levels in IgG IP. (C) Top, partial in vitro TP53 3′UTR biotinylated transcripts bearing the sites of interaction with HuR and 7SL [biot-TP53(3′)] or bearing only the sites of interaction with HuR [biot-TP53(3′Δ)]. Bottom, biotinylated RNAs were incubated with non-biotinylated 7SL and the levels of 7SL in the pulldown were measured by RT-qPCR analysis. (D) GST or GST-HuR were incubated in vitro with the biotinylated RNAs shown; following pulldown, western blot analysis was carried out to detect HuR levels. (E) GST-HuR was incubated in vitro with the biotinylated and non-biotinylated RNAs shown and its presence in the pulldown material was assessed by western blot analysis. Data in (B) are the means + S.D. from three independent experiments; data in (C) and (E) are average of two repeats showing similar results; data in (D) are representative of three repeats.

Figure 5.

Opposite regulation of p53 translation by 7SL and HuR. (A) HeLa cells were transfected with the indicated siRNAs; 48 h later, the levels of p53, HuR and loading control β-actin were assessed by western blot analysis and p53 signals were quantified by densitometry and plotted. (B) Top, luciferase reporter constructs pmirGLO-p53(3′UTR), bearing partial sequence of TP53 3′UTR (nucleotides 1421–2629) that includes 7SL and HuR binding sites, and pmirGLO-p53(3′UTRΔ) that lacks the 7SL interaction site (nucleotides 2161–2300). Thirty-six hours after transfection of the indicated siRNAs, luciferase plasmids were transfected for 6 h and followed by luciferase assay 12 h thereafter. (C) RIP analysis of HuR interaction with the FL mRNAs expressed from the reporters in panel (B); data represent enrichment levels relative to FL mRNA abundance in IgG IP, using GAPDH mRNA for normalization. (D) De novo translation of p53 as well as housekeeping control protein β-Tubulin (Tub), as assessed by ³⁵S-p53 IP and ³⁵S-Tub IP 48 h after transfection of the indicated siRNAs (details in Materials and Methods section); ‘Fold’ quantified ³⁵S-p53 signals, relative to signals in the Ctrl siRNA group. (E) and (F) Cells were transfected as described in (A) followed by fractionation through sucrose gradients. Global RNA profile for these transfection conditions are shown (E). The relative distribution of TP53 mRNA (and housekeeping GAPDH mRNA) was studied by RT-qPCR analysis of RNA in each of 10 gradient fractions (F). Data in (A)–(C) represent the means and S.D. from three independent experiments; data in (D)–(F) are representative of three independent experiments; P values are shown.

Figure 6.

Influence of 7SL silencing on cell phenotype. (A) Forty-eight hours after transfection of HeLa cells with Ctrl siRNA or 7SL siRNA, cells were subjected to FACS analysis (left) and the relative G1, S and G2/M compartments calculated (right). Data are representative of three independent experiments. (B) Measurement of [³H]-thymidine incorporation by 48 h after transfection of HeLa cells with Ctrl siRNA or 7SL siRNA. (C) β-galactosidase activity in HeLa cells 5 days after transfection with either Ctrl siRNA or 7SL siRNA. (D) Western blot analysis of the autophagy marker LC3 in 48 h after transfection of HeLa cells with Ctrl siRNA or 7SL siRNA. (E) HeLa cells were transfected with a plasmid that expresses GFP-LC3 (a fluorescent fusion protein that is recruited to autophagosomes) and with either Ctrl siRNA or 7SL siRNA; 48 h later, GFP-LC3 signals were visualized by fluorescence microscopy.